Insect resistant fertile transgenic corn plants

ABSTRACT

Fertile transgenic  Zea mays  (corn) plants which stably express recombinant DNA which is heritable are provided wherein said DNA preferably comprises a recombinant gene which encodes a seed storage protein, so that the amino acid profile of the corn is improved.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. Ser. No. 08/285,488,filed Aug. 3, 1994, now U.S. Pat. No. 5,508,468, which is a continuationof U.S. Ser. No. 07/636,089, filed Dec. 28, 1990, now abandoned, whichis a continuation-in-part of U.S. Ser. No. 07/508,045, filed Apr. 11,1990, now U.S. Pat. No. 5,484,956, which is a continuation-in-part ofU.S. Ser. No. 07/467,983, filed Jan. 22, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to fertile transgenic plants of the species Zeamays (oftentimes referred to herein as maize or corn). The inventionfurther relates to producing fertile transgenic plants via particlebombardment and subsequent selection techniques.

BACKGROUND OF THE INVENTION

Genetic engineering of plants, which entails the isolation andmanipulation of genetic material (usually in the form of DNA or RNA) andthe subsequent introduction of that genetic material into a plant orplant cells, offers considerable promise to modern agriculture and plantbreeding. Increased crop food values, higher yields, feed value, reducedproduction costs, pest resistance, stress tolerance, drought resistance,the production of pharmaceuticals, chemicals and biological molecules aswell as other beneficial traits are all potentially achievable throughgenetic engineering techniques. Once a gene has been identified, cloned,and engineered, it is still necessary to introduce it into a plant ofinterest in such a manner that the resulting plant is both fertile andcapable of passing the gene on to its progeny.

A variety of methods have been developed and are currently available forthe transformation of various plants and plant cells with DNA.Generally, these plants have been dicotyledonous, and some success hasbeen reported with certain of the monocotyledonous cereals. However,some species have heretofore proven untransformable by any method. Thus,previous to this discovery, no technology had been developed which wouldpermit the production of stably transformed Zea mays plants in which theintroduced recombinant DNA is transmitted through at least one completesexual cycle. This failure in the art is well documented in theliterature and has been discussed in a number of recent reviews (I.Potrykus, Trends in Biotechnology, 7, 269 (1989); K. Weising et al.,Ann. Rev. of Genetics, 22, 421 (1988); F. Cocking et al., Science, 236,1259 (1987)).

Some of the techniques attempted, or proposed, for introducing DNA intocorn cells include electroporation, microinjection, microprojectilebombardment, liposome fusion, Agrobacterium-mediated transfer,macroinjection, and exposure to naked DNA in solution.

For example, J. DeWet et al., Experimental Manipulation of Ovule Tissue,G. Chapman et al., eds., Longman, Inc., New York (1985) at pages 197-269and Y. Ohta, PNAS USA, 83, 715 (1986) reported the introduction of DNAinto maize by mixing pollen grains with DNA solutions, and applying thepollen to maize silks. In these papers, there is no molecular dataconfirming the introduction of the exogenous DNA into the corn cells.Arntzen et al. in published European Patent Application No. 275,069describe the incubation of DNA with maize pollen followed by pollinationof maize ears and formation of seeds. The plants derived from theseseeds were reported to contain the introduced DNA, but there is nosuggestion that the introduced DNA was transmitted through a completesexual cycle.

A. Graves et al., Plant Mol. Biol., 3, 43 (1986) reportedAgrobacterium-mediated transformation of Zea mays seedlings. Theevidence was based upon assays which can sometimes be unreliable. Todate, there have been no further reported successes with pollen andAgrobacterium-mediated transfer techniques.

Microprojectile bombardment has been reported to yield transformed corncells. The technique is disclosed in Sanford et al., Part. Sci. &Techn., 5, 27 (1987) as well as in published European patent applicationnumber 331,855 of J. C. Sanford et al. which is based upon U.S. Ser. No.07/161,807, filed Feb. 29, 1988. Klein et al., Plant Physiol., 91, 440(1989) describe production of transformed corn cells, usingmicroprojectile bombardment. However, the cells used were not capable ofregeneration into plants. Thus, no protocols have been publisheddescribing the introduction of DNA by a bombardment technique intocultures of regenerable maize cells of any type. No stable introductionof a gene has been reported that results from bombardment of maizecallus followed by regeneration fertile plants and transmission of theintroduced gene through at least one sexual cycle. D. McCabe et al., inpublished European patent application No. 270,356, disclose thebombardment of maize pollen with DNA, the application of the pollen tosilks, and the formation of seeds which reportedly contain the exogenousDNA. However, there is no evidence that the DNA was transmitted througha complete sexual cycle, and no further results have been reported bythis group.

Electroporation of corn protoplasts has been reported to result intransformed cells by M. E. Fromm et al., Nature, 319, 791 (1986)although these cells did not provide regenerated plants. Electroporationof corn protoplasts was also reported by C. Rhodes et al., Science, 240,204 (1988). Although the recipient cells were transformed and, in thelatter case, were able to regenerate into plants, the plants themselveswere sterile. In addition, methods for the production of the cell lineused by Rhodes et al. were not reproducible.

A further stumbling block to the successful production of fertiletransgenic maize plants has been in selecting those few transformants insuch a manner that neither the regeneration capacity (in the case ofprotoplasts or cell cultures) nor the fertility of the transformants aredestroyed. Due to the generally low level of transformants produced by atransformation technique, some sort of selection procedure is oftennecessary. However, selection generally entails the use of some toxicagent, e.g., a herbicide or antibiotic, which may be detrimental toeither the regenerability or the resultant plant fertility.

On the other hand, it has been known that untransformed cornprotoplasts, cultured cells and callus at least can be regenerated toform mature plants and that the resulting plants are often fertile. Forexample, R. D. Shillito et al., Bio/Technology, 7, 581 (1989), and L. M.Prioli et al., Bio/Technology, 7, 589 (1989) discuss methods forproducing protoplasts from cell cultures and recovering fertile plantstherefrom. C. A. Rhodes et al., Bio/Technology, 6, 56 (1988) discloseattempts to regenerate maize plants from protoplasts isolated fromembryogenic maize cell cultures.

However, it has not been possible for the art worker to determine whichmaize tissues or cultures are appropriate recipients for exogenous DNA,e.g., contain a useful number of cells which are receptive to and whichwill stably integrate the exogenous DNA, and at the same time be a partof the germline, i.e., part of the cell lineage that leads to the nextplant generation.

The art is thus faced with a dilemma. While certain transformationtechniques have been proposed or reported to produce transformed maizecells and certain cells and tissues have been proposed to be potentialrecipients due to their ability to regenerate plants, the art has failedto find a combination of techniques which would successfully producetransformed maize plants able to transmit the introduced DNA through onecomplete sexual cycle.

It is thus an object of the present invention to produce fertile,stably-transgenic, Zea mays plants and seeds which transmit theintroduced gene to progeny. It is a further object to produce suchstably-transgenic plants and seeds by a particle bombardment and aselection process which results in a high level of viability for atleast a portion of the transformed cells. It is a further object toproduce fertile, stably-transgenic plants of other graminaceous cerealsbesides maize.

References Cited

The references listed below are incorporated by reference herein.

Armstrong, C L, et al. (1985) Planta 164:207-214

Callis, J, et al. (1987) Genes & Develop 1:1183-1200

M. Bevan et al. (1983) Nuc Acids Res 11:36a

Chu, C C, et al. (1975) Sci Sin (Peking) 18:659-668

Cocking, F, et al. (1987) Science 236:1259-1262

DeWet et al. (1985) Proc Natl Sci USA 82:7870-7873

Freeling, J C, et al. (1976) Maydica XXI:97-112

Graves, A, et al. (1986) Plant Mol Biol 7:43-50

Green, C, et al. (1975) Crop Sci 15:417-421

Green, C, et al. (1982) Maize for Biological Research,

Plant Mol. Biol. Assoc., pp 367-372

Gritz, L, et al. (1983) Gene 25:179-188

Guilley, H, et al. (1982) Cell 30:763-773

Hallauer, A R, et al. (1988) Corn and Corn Improvement, 3rd ed.,Agronomy Society of America, pp 469-564

Jefferson, R, et al. (1987) EMBO J 6:3901-3907

Kamo, K, et al. (1985) Bot Gaz 146:327-334

Klein, T, et al. (1989) Plant Physiol 91:440-444

Klein, T, et al. (1988a) Proc Natl Acad Sci USA 85:4305-9

Klein, T, et al. (1988b) Bio/Technology 6:559-563

Lu, C, et al. (1982) Theor Appl Genet 62:109-112

McCabe, D, et al. (1988) Bio/Technology 6:923-926

Murashige, T, et al. (1962) Physiol Plant 15:473-497

Neuffer, M, (1982) Maize for Biological Research, Plant Mol Biol Assoc,pp 19-30

Phillips, R, et al. (1988) Corn and Corn Improvement, 3rd ed., AgronomySociety of America, pp 345-387

Potrykus, I (1989) Trends in Biotechnology 7:269-273

Rhodes, C A, et al. (1988) Science 240:204-7

Sambrook, J, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press

Sanford, J, et al. (1987) Part Sci & Techn 5:27-37

Weising, K, et al., (1988) Ann Rev of Genetics 22:421-478

Yanisch-Perron, L, et al. (1985) Gene 33:109-119

SUMMARY OF THE INVENTION

The present invention relates to fertile transgenic Zea mays plantscontaining recombinant DNA, preferably chromosomally integratedrecombinant DNA, which is heritable by progeny thereof.

The invention further relates to all products derived from transgenicZea mays plants, plant cells, plant parts, and seeds.

The invention further relates to transgenic Zea mays seeds stablycontaining recombinant DNA and progeny which have inherited therecombinant DNA. The invention further relates to the breeding oftransgenic plants and the subsequent incorporation of recombinant DNAinto any Zea mays plant or line.

The invention further relates to a process for producing fertiletransgenic Zea mays plants containing recombinant DNA. The process isbased upon microprojectile bombardment, selection, plant regeneration,and conventional backcrossing techniques.

The invention further relates to a process for producing fertiletransformed plants of graminaceous species other than Zea mays whichhave not been reliably transformed by traditional methods such aselectroporation, Agrobacterium, injection, and previous ballistictechniques.

The invention further relates to regenerated fertile mature maize plantsobtained from transformed embryogenic tissue, transgenic seeds producedtherefrom, and R1 and subsequent generations.

A preferred embodiment of the present invention is a fertile, transgeniccorn plant that has been stably transformed with a recombinant, chimericgene which is expressed as a seed storage protein, e.g., as the 10 kDzein protein, so that the level of at least one amino acid is elevatedabove that present in the parent, nontransformed lines. Expression ofmultiple copies of said gene or over-expression thereof, cansubstantially increase the whole kernel levels of certain amino acids,such as lysine, methionine, threonine and the like. As used herein, theterm “substantially increased” means that the level of a given aminoacid is at least 10-20% above that present in the corresponding plant orplant part which has not been transformed with said seed storage proteingene.

In preferred embodiments, this invention produces the fertile,transgenic plants by means of a recombinant DNA-coated microprojectilebombardment of clumps of friable embryogenic callus, followed by acontrolled regimen for selection of transformed callus lines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a map of plasmid vector pHYGI1 utilized in Example I. FIG.1B shows the relevant part of linearized pHYGI1 encompassing the HPTcoding sequence and associated regulatory elements. The base pairnumbers start from the 5′ nucleotide in the recognition sequence for theindicated restriction enzymes, beginning with the EcoRI site at the 5′end of the CaMV 35S promoter.

FIG. 2A shows a map of plasmid vector pBII221 utilized in Example I.FIG. 2B depicts linearized pBII221, e.g., from the HindIII cleavage siteto the EcoRI cleavage site.

FIG. 3 is a Southern blot of DNA isolated from the PH1 callus line andan untransformed control callus line, and a schematic depiction of thepHYGI1 probes used in the assay.

FIG. 4 is a Southern blot of leaf DNA isolated from R0 plantsregenerated from PH1 and untransformed callus, and a schematic depictionof the pHYGI1 probes used in the assay.

FIG. 5 is a Southern blot of leaf DNA isolated from R1 progeny of PH1 R0plants and untransformed R0 plants, and a schematic depiction of thepHYGI1 probes used in the assay.

FIG. 6 is a Southern blot of DNA isolated from the PH2 callus line andan untransformed control callus line, and a schematic depiction of thepHYGI1 probes used in the assay.

FIG. 7A shows a map of plasmid vector pZ27Z10 utilized in Example II.FIG. 7B depicts linearized plasmid pZ27Z10 encompassing the z10 codingsequence and associated regulatory elements.

FIG. 8 is a schematic depiction of the location of the PCR primerswithin the chimeric z27-z10 gene.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to the production of fertiletransgenic plants and seeds of the species Zea mays and to the plants,plant tissues, and seeds derived from such transgenic plants, as well asthe subsequent progeny and products derived therefrom, preferably thosetransgenic Zea mays plants having improved food or feed value. Thetransgenic plants produced herein include all plants of this species,including field corn, popcorn, sweet corn, flint corn and dent corn.

“Transgenicit” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of recombinant DNA, which DNA has also been referred to inthe art of genetic engineering as “heterologous DNA,” “exogenous DNA” or“foreign DNA,” wherein said DNA was introduced into the genotype by aprocess of genetic engineering, or which was initially introduced intothe genotype of a parent plant by such a process and is subsequentlytransferred to later generations by sexual crosses or asexualpropagation. As used herein, “genotype” refers to the sum total ofgenetic material within a cell, either chromosomally, orextrachromosomally borne. Therefore, the term “transgenic” as usedherein does not encompass the alteration of the genotype of Zea mays byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, viral infection or spontaneousmutation.

By “heritable” is meant that the DNA is capable of transmission throughat least one complete sexual cycle of a plant, i.e., it is passed fromone plant through its gametes to its progeny plants.

The transgenic plants of this invention may be produced by (i)establishing a regenerable cell culture, preferably a friableembryogenic callus, (ii) transforming said cell culture by amicroprojectile bombardment technique, (iii) identifying or selectingtransformed cells, and (iv) regenerating fertile transgenic plants fromthe transformed cells. Some of the plants of this invention may beproduced from the transgenic seed produced from the fertile transgenicplants using conventional crossbreeding techniques to develop transgenicelite lines and varieties, or commercial hybrid seed containingrecombinant DNA.

I. Plant Lines and Tissue Cultures

The cells which have been found particularly useful to produce thefertile transgenic maize plants herein are those callus cells which areregenerable, both before and after undergoing a selection regimen asdetailed further below. Generally, these cells will be derived frommeristematic tissue which contains cells which have not yet terminallydifferentiated. Such tissue in graminaceous cereals in general and inmaize, in particular, comprise tissues found in juvenile leaf basalregions, immature tassels, immature embryos, and coleoptilar nodes.Preferably, immature embryos are used. Methods of preparing andmaintaining callus from such tissue and plant types are well known inthe art and details on so doing are available in the literature, c. f.Phillips et al. (1988), the disclosure of which is hereby incorporatedby reference.

The specific callus used must be able to regenerate into a fertileplant. The specific regeneration capacity of particular callus isimportant to the success of the bombardment/selection process usedherein because during and following selection, regeneration capacity maydecrease significantly. It is therefore important to start with culturesthat have as high a degree of regeneration capacity as possible. Calluswhich is more than about 3 months and up to about 36 months of age hasbeen found to have a sufficiently high level of regenerability and thusis preferred. The regenerative capacity of a particular culture may bereadily determined by transferring samples thereof to regenerationmedium and monitoring the formation of shoots, roots, and plantlets. Therelative number of plantlets arising per petri dish or per gram freshweight of tissue may be used as a rough quantitative estimate ofregeneration capacity. Generally, a culture which will produce at leastone plant per gram of callus tissue is preferred.

While maize callus cultures can be initiated from a number of differentplant tissues, the cultures useful herein are preferably derived fromimmature maize embryos which are removed from the kernels of an ear whenthe embryos are about 1-3 mm in length. This length generally occursabout 9-14 days after pollination. Under aseptic conditions, the embryosare placed on conventional solid media with the embryo axis down(scutellum up). Callus tissue appears from the scutellum after severaldays to a few weeks. After the callus has grown sufficiently, the cellproliferations from the scutellum may be evaluated for friableconsistency and the presence of well-defined embryos. By “friableconsistency” it is meant that the tissue is easily dispersed withoutcausing injury to the cells. Tissue with this morphology is thentransferred to fresh media and subcultured on a routine basis aboutevery two weeks. Sieving to reduce clumping and/or to increase cellsurface area may be employed during the subculturing period.

The callus initiation media is preferably solid. In preferredembodiments, the initiation/maintenance media is typically based on theN6 salts of Chu et al. (1975) as described in Armstrong et al. (1985) orthe MS salts of Murashige et al. (1962). The basal medium issupplemented with sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D).Supplements such as L-proline and casein hydrolysate have been found toimprove the frequency of initiation of callus cultures, morphology, andgrowth. The cultures are generally maintained in the dark, though lowlight levels may also be used. The level of synthetic hormone 2,4-D,necessary for maintenance and propagation, should be generally about 0.3to 3.0 mg/l.

Although successful transformation and regeneration has beenaccomplished herein with friable embryogenic callus, this is not meantto imply that other transformable regenerable cells, tissue, or organscannot be employed to produce the fertile transgenic plants of thisinvention. The only actual requirement for the cells which aretransformed is that after transformation they must be capable ofregeneration of a plant containing the recombinant DNA following theparticular selection or screening procedure actually used.

For example, cells grown in liquid suspension culture may be used. Toestablish these cultures, the type II callus, after 4-6 months, istransferred to liquid growth media. Methods and references for theproduction of regenerable suspension cell cultures are given by C. E.Green et al. in Maize for Biological Research, Plant Molec. Biol. Assoc.(1982) at pages 367-372, R. Phillips et al., Corn and Corn Improvement,Agronomy Soc. Amer., (3d ed., 1988) at pages 345-387, and I. Vasil, CellCulture and Somatic Cell Genetics of Plants, Vol. I, LaboratoryProcedures and Their Applications, Academic Press (1984) at pages152-158. Typically, the liquid growth media for suspension cultures isof similar formulation to the solid callus induction media. ABA(abscisic acid) (10⁻⁷M) may be added to the liquid growth media toaugment regenerative capacity and enhance culture vitality. It ispreferred that the callus not be sieved prior to introduction into theliquid media.

The cultures in liquid media are subcultured as appropriate formaintaining active growth and their regenerative properties. Inpreferred embodiments, the cultures are subcultured once a week at a1:8-9 dilution with fresh growth medium.

II. DNA Used for Transformation

As used herein, the term “recombinant DNA” refers to DNA that has beenderived or isolated from any source, that may be subsequently chemicallyaltered, and later introduced into Zea mays. An example of recombinantDNA “derived” from a source, would be a DNA sequence that is identifiedas a useful fragment within a given organism, and which is thenchemically synthesized in essentially pure form. An example of such DNA“isolated” from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, e.g., by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering.

Therefore “recombinant DNA” includes completely synthetic DNA,semi-synthetic DNA, DNA isolated from biological sources, and DNAderived from introduced RNA. Generally, the recombinant DNA is notoriginally resident in the Zea mays genotype which is the recipient ofthe DNA, but it is within the scope of the invention to isolate a genefrom a given Zea mays genotype, and to subsequently introduce multiplecopies of the gene into the same genotype, e.g., to enhance productionof a given gene product such as a storage protein.

The recombinant DNA includes but is not limited to, DNA from plantgenes, and non-plant genes such as those from bacteria, yeasts, animalsor viruses; modified genes, portions of genes, chimeric genes, includinggenes from the same or different Zea mays genotype.

The recombinant DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, that can also contain codingregions flanked by regulatory sequences which promote the expression ofthe recombinant DNA present in the resultant corn plant. For example,the recombinant DNA may itself comprise or consist of a promoter that isactive in Zea mays, or may utilize a promoter already present in the Zeamays genotype that is the transformation target.

The compositions of, and methods for, constructing recombinant DNA whichcan transform certain plants are well known to those skilled in the art,and the same compositions and methods of construction may be utilized toproduce the DNA useful herein. The specific composition of the DNA isnot central to the present invention and the invention is not dependentupon the composition of the specific recombinant DNA used. K. Weising etal., Ann. Rev. Genetics, 22, 421 (1988) describes suitable DNAcomponents, selectable marker genes, reporter genes, enhancers, introns,and the like, as well as provides suitable references for compositionstherefrom. J. Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press (2d ed., 1989), provides suitablemethods of construction. Generally, the recombinant DNA will berelatively small, i.e., less than about 30 kb to minimize anysusceptibility to physical, chemical, or enzymatic degradation which isknown to increase as the size of the DNA increases.

Suitable recombinant DNA for use herein includes all DNA which providesfor, or enhances, a beneficial feature of the resultant transgenic cornplant. The DNA may encode proteins or antisense RNA transcripts in orderto promote increased food values, higher yields, pest resistance,disease resistance, herbicide resistance, and the like. For example, theDNA can encode a DHDP synthase, as does the dap A gene, for increasedlysine production; Bacillus thuringiensis (Bt), δ-endotoxin or aprotease inhibitor for insect resistance; bacterial EPSP synthase forresistance to glyphosate herbicide; and chitinase or glucanendo-1,3-β-glucosidase for fungicidal properties.

Of importance in improving food or feed value are genes encodingproteins that contain high levels of essential amino acids. For example,to be nutritionally adequate and support optimal growth of chickens,corn-soybean meal poultry feed is generally supplemented with syntheticmethionine or a methionine analog. The development of lines of cornwhich supply higher levels of methionine can reduce the need formethionine supplements. The development of such high methionine cornlines can be accomplished by introducing into the corn genome a highlyexpressed gene or genes encoding a high-methionine protein.

Examples of genes encoding high-methionine proteins include: 1.) thegene encoding a maize 15 kD-zein protein (11% methionine), (Pedersen etal., J. Biol. Chem., 261, 6279 (1986)); 2.) the gene encoding a Brazilnut storage protein (18% methionine), (Altenbach et al., Plant Mol.Biol., 8; 239 (1987)) and 3.) the gene encoding a maize 10 kD-zeinprotein (22.5% methionine), (Kirihara et al., Gene, 71, 359 (1988)). Thepreferred gene is the 10 kD-zein gene, since it is an endogenous maizegene whose protein product normally accumulates in the kernel and istwice as high in methionine than the 15 kD-zein protein. To obtain highlevels of expression in the seed, its coding sequence optionally may befused to the regulatory sequence from a highly-expressed, seed-specificgene. Alternatively, introduction of additional copies of the intactendogenous 10 kD-zein gene into the corn genome can also increase themethionine content of corn seed.

Lysine, an amino acid essential in the diets of humans and monogastricanimals, is among the three most limiting amino acids in most of thestaple crops, the cereals in particular. Consequently, grain-based dietsmust be supplemented with synthetic lysine or with lysine-containingoilseed protein meals. Further, since most oilseed meals are themselvesinadequate lysine sources, balancing the feed mixture for lysinefrequently results in meals which are too high in other, less desirablenutrients. Therefore, a method to increase the lysine content of eitherthe cereal grains or the oilseed crops or both would result insignificant added nutritional value, as well as a significant costsavings to end-users such as the swine and poultry producers.

One approach to improving the lysine content of cereals is to deregulatethe biosynthetic pathway, allowing free lysine to accumulate. The dap Agene of Escherichia coli encodes dihydrodipicolinic acid synthase(DHDPS), a key regulatory enzyme whose activity in plants is stronglyfeedback-inhibited by lysine. The bacterial enzyme is about 200-foldless sensitive to inhibition by lysine. The introduction and expressionof the dap A gene in plant cells would allow the synthesis of freelysine to continue after the native plant DHDPS has been completelyinhibited.

Of particular importance in maintaining yield from corn plants andcontributing significantly to controlling the cost of growing a corncrop is the protection of the corn against attack by insect pests. Inthe USA the major insect pests of corn include a variety of Lepidopterapests such as the European corn borer, cutworms and earworms as well asColeoptera species such as Diabrotica spp. The protection of cornagainst insect attack is expensive to the grower and requires the use oftoxic chemical insecticides applied in a timely manner. Sincetraditional methods of breeding and selection have not allowed for thedevelopment of new lines of corn that are substantially resistant tomajor insect pests, the introduction and inheritance of insectresistance genes or sequences in corn plants in accord with the presentinvention would reduce costs to the grower, reduce the use of toxicchemical insecticides and provide for more effective control of insectpests.

Essential components of the present invention are the introduction ofthe insect resistance gene into the corr sell, mitotic replication ofthe gene so that the gene is incorporated into whole corn plants and isultimately inherited by subsequent offspring of the plant via theprocesses of mitotic and meiotic division.

Bacillus thuringiensis (or “Bt”) bacteria include nearly 20 knownsubspecies of bacteria which produce endotoxin polypeptides that aretoxic when ingested by a wide variety of insect species. The biology andmolecular biology of the endotoxin proteins (Bt proteins) andcorresponding genes (Bt genes) has been reviewed recently by H. R.Whitely et al., Ann. Rev. Microbiol., 40, 549 (1986) and by H. Hofte etal., Microbiol. Rev., 53, 242 (1989). Genes coding for a variety of Btproteins have been cloned and sequenced. Research has demonstrated thata segment of the Bt polypeptide is essential for toxicity to a varietyof Lepidoptera pests and is contained within approximately the first 50%of the Bt polypeptide molecule. Consequently, a truncated Bt polypeptidecoded by a truncated Bt gene will in many cases retain its toxicitytowards a number of Lepidoptera insect pests. The HD73 and HD1 Btpolypeptides have been shown to be toxic to the larvae of the importantLepidoptera insect pests of corn plants in the USA such as the Europeancorn borer, cutworms and earworms. The genes coding for the HD1 and HD73Bt polypeptides have been cloned and sequenced by M. Geiser et al.,Gene, 48, 109 (1986) and M. J. Adang et al., Gene, 36, 289 (1985),respectively, and can be cloned from HD1 and HD73 strains obtained fromculture collections (e.g. Bacillus Genetic Stock Center, Columbus, Ohioor USDA Bt stock collection Peoria, Ill.) using standard protocols.

DNA coding for new, previously uncharacterized Bt toxins, may be clonedfrom the host Bacillus organism using protocols that have previouslybeen used to clone Bt genes. These include the construction of a bank ofDNA isolated from the Bacillus organism in a suitable plasmid or phagevector replicated in a suitable host and the use of anti-bodies, raisedagainst the Bt protein or DNA isolated from a homologous Bt genesequence, to identify transformants that contain the cloned Bt sequence.The approximate location of the Bt coding sequence may be initiallydetermined using deletion analysis of the cloned DNA. The preciselocation of the Bt coding sequence could be determined using a varietyof standard methods, including the determination of the sequence of thecloned segment of DNA, determining the presence of large open readingframes in this sequence that could code for the Bt protein andconfirmation of the Bt coding nature of the DNA sequence by comparingthe amino acid sequence derived from the DNA sequence with that derivedfrom partial amino acid sequencing of the Bt protein.

A chimeric Bt gene useful in the present invention would comprise a 5′DNA sequence, comprising a sequence of DNA which will allow for theinitiation of transcription (“promoter”) and translation of a downstreamlocated Bt sequence in a corn plant. The chimeric Bt gene would alsocomprise a 3′ DNA sequence that includes a sequence derived from the 3′non-coding region of a gene that can be expressed in corn. Mostimportantly, the chimeric Bt gene will include a DNA sequence coding fora toxic Bt polypeptide produced by Bacillus thuringiensis or toxicportions thereof or having substantial amino sequence homology thereto.The Bt coding sequence would include: (i) DNA sequences which code forinsecticidally active proteins that have substantial homology to Btendotoxins that are active against insect pests of corn, e.g., the HD73or HD1 Bt sequences; (ii) sequences coding for insecticidally activesegments of the Bt endotoxin polypeptide, e.g., insecticidally activeHD73 or HD1 polypeptides truncated from the carboxy and/or aminotermini; (iii) a truncated Bt sequence fused in frame with a sequence(s)that codes for a polypeptide that provides some additional advantagesuch as: (a) genes that are selectable, e.g., genes that conferresistance to antibiotics or herbicides, (b) reporter genes whoseproducts are easy to detect or assay, e.g., luciferase orbeta-glucuronidase; (c) DNA sequences that code for polypeptidesequences that have some additional use in stabilizing the Bt proteinagainst degradation or enhance the efficacy of the Bt protein againstinsects, e.g., protease inhibitors and (d) sequences that help directthe Bt protein to a specific compartment inside or outside the corncell, e.g., a signal sequence.

To obtain optimum synthesis of the Bt protein in corn, it may also beappropriate to adjust the DNA sequence of the Bt gene to more resemblethe genes that are efficiently expressed in corn. Since the codon usageof many Bt genes, including the HD73 and HD1 genes, is more similar tothat used by Bacillus species and dissimilar to that used by genes thatare expressed in maize, the expression of the Bt gene in maize cells canbe improved by the replacement of these rarely used Bacillus codons withthose that are used more frequently in maize plants (See E. Murray etal., Nucl. Acids Res., 17, 477 (1989)). Such replacement of codons wouldrequire the substitution of bases without changing the amino acidsequence of the resulting Bt polypeptide. The Bt polypeptide would beidentical in sequence to the bacterial polypeptide or segments thereof.The complete Bt coding sequence, or sections thereof, containing ahigher proportion of maize preferred codons than the original bacterialgene could be synthesized using standard chemical synthesis protocols,and introduced or assembled into the Bt gene using standard protocols,such as site-directed mutagenesis or DNA polymerization and ligation andthe like.

In general, the plant codon usage pattern of plants more closelyresembles that of man and other higher ekaryotes that unicellularorganisms, close to the overall preference for G+C content in codonposition III. Also in general, the most important factor indiscriminating between monocot and dicot patterns of codon usage is thepercentage G+C content of the degenerate third base. In monocots, 16 of18 amino acids favor G+C in this position, while dicots only favor G+Cin 7 of 18 amino acids.

Aside from recombinant DNA sequences that serve as transcription unitsor portions thereof, useful recombinant DNA may be untranscribed,serving a regulatory or a structural function. Also, the DNA may beintroduced to act as a genetic tool to generate mutants and/or assist inthe identification, genetic tagging, or isolation of segments of cornDNA. Additional examples may be found in Weising, cited supra.

The recombinant DNA to be introduced into the plant cells further willgenerally contain either a selectable marker or a reporter gene or bothto facilitate identification and selection of transformed cells.Alternatively, the selectable marker may be carried on a separate pieceof DNA and used in a co-transformation procedure. Both selectablemarkers and reporter genes may be flanked with appropriate regulatorysequences to enable expression in plants. Useful selectable markers arewell known in the art and include, for example, antibiotic and herbicideresistance genes.

Specific examples of selectable marker genes are disclosed in Weising etal., cited supra. A preferred selectable marker gene is the hygromycinphosphotransferase (HPT) coding sequence, which may be derived from E.coli and which confers resistance to the antibiotic hygromycin B. Otherselectable markers include the aminoglycoside phosphotransferase gene oftransposon Tn5 (AphII) which encodes resistance to the antibioticskanamycin, neomycin and G418, as well as those genes which code forresistance or tolerance to glyphosate, 2,2-dichloropropionic acid,methotrexate, imidazolinone herbicides, sulfonylurea herbicides,bromoxynil, phosphinothricin and other herbicidal compounds. Thoseselectable marker genes which confer resistance or tolerance to thesephytotoxic compounds are also of commercial utility in the resultingtransformed plants. Selectable marker genes encoding enzymes whichimpart resistance to phytotoxic compounds are listed in Table 1, below.

TABLE 1 Selectable Marker Genes Resistance Confers Gene or EnzymeResistance to: Reference Neomycin phospho- G-418, neomycin, P. J.Southern et al., J. transferase (neo) kanamycin Mol. Appl. Gen., 1, 327(1982) Hygromycin phos- Hygromycin B Y. Shimizu et al., Mol.photranferase (hpt Cell Biol., 6, 1074 (1986) or hyg) DihydrofolateMethotrexate W. W. Kwok et al., PNAS reductase (dhfr) USA, 4552 (1986)Phosphinothricin Phosphinothricin M. DeBlock et al., acetyltransferaseEMBO J., 6, 2513 (1987) (bar) 2,2-Dichloropro- 2,2-Dichloropro- V.Buchanan-Wollaston et pionic acid pionic acid al., J. Cell. Biochem.,dehalogenase (Dalapon) Supp. 13D, 330 (1989) AcetohydroxyacidSulfonylurea, P. C. Anderson et al. (U.S. synthase imidazolinone andPat. No. 4,761,373); G. W. triazolopyrimidine Haughn et al., Mol. Gen.herbicides Genet., 211, 266 (1988) 5-Enolpyruvyl- Glyphosate L. Comai etal., Nature, shikimate-phosphate 317, 741 (1985) synthase (aroA)Haloarylnitrilase Bromoxynil D. M. Stalker et al., published PCT appln.WO87/04181 Acetyl-coenzyme A Sethoxydim, W. B. Parker et al., Plantcarboxylase haloxyfop Physiol., 92, 1220 (1990) DihydropteroateSulfonamide F. Guerineau et al., Plant synthase (sul I) herbicidesMolec. Biol., 15, 127 (1990) 32 kD photosystem Triazine herbicides J.Hirschberg et al., II polypeptide Science, 222, 1346 (1983) (psbA)Anthranilate 5-Methyltryptophan K. Hibberd et al., (U.S. synthase Pat.No. 4,581,847) Dihydrodipicolinic Aminoethyl cysteine K. Glassman etal., acid synthase published PCT application (dap A) No. WO89/11789

Reporter genes are used for identifying potentially transformed cellsand for evaluating the functionality of regulatory sequences. Reportergenes which encode for easily assayable marker proteins are well knownin the art. In general, a reporter gene is a gene which is not presentin or expressed by the recipient organism or tissue and which encodes aprotein whose expression is manifested by some easily detectableproperty, e.g., phenotypic change or enzymatic activity. Examples ofsuch genes are provided in weising et al., supra. Preferred genesinclude the chloramphenicol acetyl transferase gene (cat) from Tn9 of E.coli, the beta-glucuronidase gene (gus) of the uida locus of E. coli,and the luciferase gene from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells. A preferred such assay entails theuse of the E. coli beta-glucuronidase (GUS) gene (R. Jefferson et al.,EMBO J., 16, 3901 (1987)). Maize cells transformed and expressing thisgene will stain blue upon exposure to the substrate,5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GLUC), in theextracellular medium.

The regulatory sequences useful herein include any constitutive,inducible, tissue or organ specific, or developmental stage specificpromoter which can be expressed in the particular plant cell. Suitablesuch promoters are disclosed in Weising et al., supra. The following isa partial representative list of promoters suitable for use herein:regulatory sequences from the T-DNA of Agrobacterium tumefaciens,including mannopine synthase, nopaline synthase, and octopine synthase;alcohol dehydrogenase promoter from corn; light-inducible promoters suchas the ribulose-bisphosphate-carboxylase/oxygenase small subunit genesfrom a variety of species; and the major chlorophyll a/b binding proteingene promoters; 35S and 19S promoters of cauliflower mosaic virus(CaMV); developmentally regulated promoters such as the waxy, zein, orbronze promoters from maize; as well as synthetic or other naturalpromoters which are either inducible or constitutive, including thosepromoters exhibiting organ-specific expression or expression at specificdevelopment stage(s) of the plant.

Other elements such as introns, enhancers, polyadenylation sequences andthe like, may also be a part of the recombinant DNA. Such elements mayor may not be necessary for the function of the DNA, but may provideimproved expression of the DNA by affecting transcription, stability ofthe mRNA, or the like. Such elements may be included in the DNA asdesired to obtain the optimal performance of the transforming DNA in theplant. For example, the maize AdhIS first intron may be placed betweenthe promoter and the coding sequence in a particular recombinant DNAconstruction. This intron, when included in a DNA construction, is knownto increase production of a protein in maize cells. (J. Callis et al.,Genes and Develop., 1, 1183 (1987)). However, sufficient expression fora selectable marker to perform satisfactorily can often be obtainedwithout an intron. (T. Klein et al., Plant Physiol., 91, 440 (1989)). Anexample of an alternative suitable intron is the shrunken-1 first intronof Zea mays. These other elements must be compatible with the remainderof the DNA constructions.

III. DNA Delivery Process

The recombinant DNA can be introduced into the regenerable maize cellcultures, preferably into callus cultures via a particle bombardmentprocess. A general description of a suitable particle bombardmentinstrument is provided in Sanford et al. (1987), the disclosure of whichis incorporated herein by reference. While protocols for the use of theinstrument in the bombardment of maize non-regenerable suspensionculture cells are described in Klein et al. (1988a, 1988b, and 1989), noprotocols have been published for the bombardment of callus cultures orregenerable maize cells.

In a microprojectile bombardment process, also referred to as abiolistic process, the transport of the recombinant DNA into the callusis mediated by very small particles of a biologically inert material.When the inert particles are coated with DNA and accelerated to asuitable velocity, one or more of the particles is able to enter intoone or more of the cells where the DNA is released from the particle andexpressed within the cell. Some of the recipient cells stably retain theintroduced DNA and express it.

The particles, called microprojectiles, are generally of a high densitymaterial such as tungsten or gold. They are coated with the DNA ofinterest. The microprojectiles are then placed onto the surface of amacroprojectile which serves to transfer the motive force from asuitable energy source to the microprojectiles. After themacroprojectile and the microprojectiles are accelerated to the propervelocity, they contact a blocking device which prevents themacroprojectile from continuing its forward path but allows theDNA-coated microprojectiles to continue on and impact the recipientcallus cells. Suitable such instruments may use a variety of motiveforces such as gunpowder or shock waves from an electric arc discharge(P. Christou et al., Plant Physiol., 87, 671 (1988)). An instrument inwhich gunpowder is the motive force is currently preferred and such isdescribed and further explained in Sanford et al. (1987), the disclosureof which is incorporated herein by reference.

A protocol for the use of the gunpowder instrument is provided in Kleinet al. (1988a,b) and involves two major steps. First, tungstenmicroprojectiles are mixed with the DNA, calcium chloride, andspermidine in a specified order in an aqueous solution. Theconcentrations of the various components may be varied as taught. Thepreferred procedure entails exactly the procedure of Klein et al.(1988b) except for doubling the stated optimum DNA concentration.Secondly, the DNA-coated microprojectiles, macroprojectiles, andrecipient cells are placed in position in the instrument and the motiveforce is applied to the macroprojectiles. Parts of this step which maybe varied include the distance of the recipient cells from the end ofthe barrel as well as the vacuum level in the sample chamber. Therecipient tissue is positioned 2-15, preferably 5 cm below the stoppingplate tray.

The callus cultures useful herein for generation of transgenic plantsshould generally be about midway between transfer periods, and thus,past any “lag” phase that might be associated with a transfer to a newmedia, but also before reaching any “stationary” phase associated withan extended period of time without subculture to fresh media. The tissuemay be used in the form of pieces of about 30 to 80, preferably about 40to 60, mg. The clumps can be placed on a petri dish or other surface andarranged in essentially any manner, recognizing that (i) the space inthe center of the dish will receive the heaviest concentration ofmetal-DNA particles and the tissue located there is likely to sufferdamage during bombardment and, (ii) the number of particles reaching thetissue will decrease with increasing distance of the tissue from thecenter of the blast so that the tissue far from the center of the dishis less likely to be bombarded. A mesh screen, preferably of metal, maybe laid on the dish to prevent splashing or ejection of the tissue. Analternative method for presentation of the tissue for bombardment is tospread the tissue onto a filter paper in a thin layer. The tissue may bebombarded one or more times with the DNA-coated metal particles.

IV. Selection Process

Once the calli have been bombarded with the recombinant DNA and the DNAhas penetrated some of the cells, it is necessary to identify and selectthose cells which both contain the recombinant DNA and still retainsufficient regenerative capacity. There are two general approaches whichhave been found useful for accomplishing this. First, the transformedcalli or plants regenerated therefrom can be screened for the presenceof the recombinant DNA by various standard methods which could includeassays for the expression of reporter genes or assessment of phenotypiceffects of the recombinant DNA, if any. Alternatively, and preferably,when a selectable marker gene has been transmitted along with or as partof the recombinant DNA, those cells of the callus which have beentransformed can be identified by the use of a selective agent to detectexpression of the selectable marker gene.

Selection of the putative transformants is a critical part of thesuccessful transformation process since selection conditions must bechosen so as to allow growth and accumulation of the transformed cellswhile simultaneously inhibiting the growth of the non-transformed cells.The situation is complicated by the fact that the vitality of individualcells in a population is often highly dependent on the vitality ofneighboring cells. Also, the selection conditions must not be so severethat the plant regeneration capacity of the callus cells and thefertility of the resulting plant are precluded. Thus, the effects of theselection agent on cell viability and morphology should be evaluated.This may be accomplished by experimentally producing a growth inhibitioncurve for the given selective agent and tissue being transformedbeforehand. This will establish the concentration range which willinhibit growth.

When a selectable marker gene has been used, the callus clumps may beeither allowed to recover from the bombardment on non-selective media,or preferably, directly transferred to media containing the selectionagent.

Selection procedures involve exposure to a toxic agent and may employsequential changes in the concentration of the agent and multiple roundsof selection. The particular concentrations and cycle lengths are likelyto need to be varied for each particular agent. A currently preferredselection procedure entails using an initial selection round at arelatively low toxic agent concentration and then later round(s) athigher concentration(s). This allows the selective agent to exert itstoxic effect slowly over a longer period of time. Preferably, theconcentration of the agent is initially such that about a 5-40% level ofgrowth inhibition will occur, as determined from a growth inhibitioncurve. The effect may be to allow the transformed cells topreferentially grow and divide while inhibiting untransformed cells, butnot to the extent that growth of the untransformed cells is prevented.Once the few individual transformed cells have divided a sufficientnumber of times, the tissue may be shifted to media containing a higherconcentration of the toxic agent to kill essentially all untransformedcells. The shift to the higher concentration also reduces thepossibility of non-transformed cells adapting to the agent. The higherlevel is preferably in the range of about 30 to 100% growth inhibition.The length of the first selection cycle may be from about 1 to 4 weeks,preferably about 2 weeks. Later selection cycles may be from about 1 to12 weeks, preferably about 2 to 10 weeks. Putative maize transformantscan generally be identified as proliferating sectors of tissue among abackground of non-proliferating cells. The callus may also be culturedon non-selective media at various times during the overall selectionprocedure.

Once a callus sector is identified as a putative transformant,transformation can be confirmed by phenotypic and/or genotypic analysis.If a selection agent is used, an example of phenotypic analysis is tomeasure the increase in fresh weight of the putative transformant ascompared to a control on various levels of the selective agent. Otheranalyses that may be employed will depend on the function of therecombinant DNA. For example, if an enzyme or protein is encoded by theDNA, enzymatic or immunological assays specific for the particularenzyme or protein may be used. Other gene products may be assayed byusing a suitable bioassay or chemical assay. Other such techniques arewell known in the art and are not repeated here. The presence of thegene can also be confirmed by conventional procedures, i.e., Southernblot or polymerase chain reaction (PCR) or the like.

V. Regeneration of Plants and Production of Seed

Cell lines which have been shown to be transformed must then beregenerated into plants and the fertility of the resultant plantsdetermined. Transformed lines which test positive by genotypic and/orphenotypic analysis are then placed on a medium which promotes tissuedifferentiation and plant regeneration. Regeneration may be carried outin accordance with standard procedures well known in the art. Theprocedures commonly entail reducing the level of auxin whichdiscontinues proliferation of a callus and promotes somatic embryodevelopment or other tissue differentiation. One example of such aregeneration procedure is described in Green et al. (1982). The plantsare grown to maturity in a growth room or greenhouse and appropriatesexual crosses are made as described by Neuffer (1982).

Regeneration, while important to the present invention, may be performedin any conventional manner. If a selectable marker has been transformedinto the cells, the selection agent may be incorporated into theregeneration media to further confirm that the regenerated plantlets aretransformed. Since regeneration techniques are well known and notcritical to the present invention, any technique which accomplishes theregeneration and produces fertile plants may be used.

VI. Analysis of R1 Progeny

The plants regenerated from the transformed callus are referred to asthe R0 generation or R0 plants. The seeds produced by various sexualcrosses of the R0 generation plants are referred to as R1 progeny or theR1 generation. When R1 seeds are germinated, the resulting plants arealso referred to as the R1 generation.

To confirm the successful transmission and inheritance of therecombinant DNA through one complete sexual cycle, the R1 generationshould be analyzed to confirm the presence of the transforming DNA. Theanalysis may be performed in any of the manners such as were disclosedabove for analyzing the bombarded callus for evidence of transformation,taking into account the fact that plants and plant parts are being usedin place of the callus.

The recombinant DNA can show different types of inheritance patterns fordifferent transformed lines. For example, the recombinant DNA may beinherited in the progeny according to the rules of Mendelianinheritance. This type of heritability involves transmission of the DNAthrough both the male and female gametes and is associated with stableintegration or incorporation into maize nuclear DNA. An alternative typeof inheritance pattern is maternal inheritance, in which the recombinantDNA is transmitted primarily or exclusively through the female gametes.The inheritance pattern for a particular transformant may be ascertainedby analysis of the progeny of various sexual crosses.

VII. Establishment of the Recombinant DNA in Other Maize Varieties

Fertile, transgenic plants may then be used in a conventional maizebreeding program in order to incorporate the introduced recombinant DNAinto the desired lines or varieties. Methods and references forconvergent improvement of corn are given by Hallauer et al., (1988)incorporated herein by reference. Among the approaches that conventionalbreeding programs employ is a conversion process (backcrossing).Briefly, conversion is performed by crossing the initial transgenicfertile plant to elite inbred lines. The progeny from this cross willsegregate such that some of the plants will carry the recombinant DNAwhereas some will not. The plants that do carry the DNA are then crossedagain to the elite inbred lines resulting in progeny which segregateonce more. This backcrossing process is repeated until the originalelite inbred has been converted to a line containing the recombinantDNA, yet possessing all important attributes originally found in theparent. Generally, this will require about 6-8 generations. A separatebackcrossing program will be generally used for every elite line that isto be converted to a genetically engineered elite line.

Generally, the commercial value of the transformed corn produced hereinwill be greatest if the recombinant DNA can be incorporated into manydifferent hybrid combinations. A farmer typically grows several hybridsbased on differences in maturity, standability, and other agronomictraits. Also, the farmer must select a hybrid based upon his or hergeographic location since hybrids adapted to one region are generallynot adapted to another because of differences in such traits asmaturity, disease, and insect resistance. As such, it is necessary toincorporate the recombinant DNA into a large number of parental lines sothat many hybrid combinations can be produced containing the desirableheterologous DNA.

Corn breeding and the techniques and skills required to transfer genesfrom one line or variety to another are well known to those skilled inthe art. Thus, introducing recombinant DNA into any other line orvariety can be accomplished by these breeding procedures.

VIII. Uses of Transgenic Plants

The transgenic plants produced herein are expected to be useful for avariety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as pest resistance, herbicideresistance or increased yield), beneficial to the consumer of the grainharvested from the plant (e.g., improved nutritive content in human foodor animal feed), or beneficial to the food processor (e.g., improvedprocessing traits). In such uses, the plants are generally grown for theuse of their grain in human or animal foods. However, other parts of theplants, including stalks, husks, vegetative parts, and the like, mayalso have utility, including use as part of animal silage or forornamental purposes. Often, chemical constituents (e.g., oils orstarches) of corn and other crops are extracted for foods or industrialuse and transgenic plants may be created which have enhanced or modifiedlevels of such components.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules.

The transgenic plants may also be used in commercial breeding programs,or may be crossed or bred to plants of related crop species.Improvements encoded by the recombinant DNA may be transferred, e.g.,from corn cells to cells of other species, e.g., by protoplast fusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences which can be used to identify proprietary linesor varieties.

The following non-limiting examples are illustrative of the presentinvention. They are presented to better explain the general procedureswhich were used to prepare the fertile Zea mays plants of this inventionwhich stably express the recombinant DNA and which transmit that DNA toprogeny. All parts and percentages are by weight unless otherwisespecified. It must be recognized that the probability of a specifictransformation event occurring is a function of the amount of materialsubjected to the transformation procedure. Thus, when individualsituations arise in which the procedures described herein do not producea transformed product, repetition of the Procedures will be required.

Example I Fertile Transgenic Zea Mays Plants

I. Initiation and Maintenance of Maize Cell Cultures which Retain PlantRegeneration Capacity

Friable, embryogenic maize callus cultures were initiated from hybridimmature embryos produced by pollination of inbred line A188 plants(University of Minnesota, Crop Improvement Association) with pollen ofinbred line B73 plants (Iowa State University). Ears were harvested whenthe embryos had reached a length of 1.5 to 2.0 mm. Each ear was surfacesterilized in 50% v/v commercial bleach (2.63% w/v sodium hypochlorite)for 20 min. at room temperature. The ears were then washed with sterile,distilled, deionized water. Immature embryos were aseptically isolatedand placed on nutrient initiation/maintenance medium with the root/shootaxis exposed to the medium. Initiation/maintenance medium (hereinafterreferred to as “F medium”) consisted of N6 basal media (Chu 1975) with2% (w/v) sucrose, 1.5 mg per liter 2,4-dichlorophenoxyacetic acid(2,4-D), 6 mM proline, and 0.25% Gelrite (Kelco, Inc., San Diego). ThepH was adjusted to 5.8 prior to autoclaving. Unless otherwise stated,all tissue culture manipulations were carried out under sterileconditions.

The immature embryos were incubated at 26° C. in the dark. Cellproliferations from the scutellum of the immature embryos were evaluatedfor friable consistency and the presence of well-defined somaticembryos. Tissue with this morphology was transferred to fresh media 10to 14 days after the initial plating of the immature embryos. The tissuewas then subcultured on a routine basis every 14 to 21 days. Sixty toeighty milligram quantities of tissue were removed from pieces of tissuethat had reached a size of approximately one gram and were transferredto fresh media. Subculturing always involved careful visual monitoringto be sure that only tissue of the correct morphology was maintained.The presence of somatic embryos ensured that the cultures would giverise to plants under the proper conditions. The cell culture named AB12used in this example was such a culture and had been initiated about 1year before bombardment.

II. Plasmids

The plasmid pHYGI1, was constructed in the vector pBS+ (Stratagene,Inc., San Diego, Calif.), a 3.2 Kb circular plasmid, using standardrecombinant DNA techniques. The 553 bp Bcl-BamHI fragment containing themaize AdhIS first intron (Callis et al. 1987) was inserted between theCaMV 35S promoter and the hygromycin coding sequence of pCHN1-1, aplasmid constructed in accord with Example V. A map of pHYGI1 isprovided as FIG. 1. A sample of pHYGI1 was deposited at the AmericanType Culture Collection, Rockville, Md., USA, on Mar. 16, 1990, underthe provisions of the Budapest Treaty, and assigned accession number40774.

pBII221 contains the E. coli β-glucuronidase coding sequence flanked atthe 5′ end by the CaMV 35S promoter and at the 3′ end by the nospolyadenylation sequence. The plasmid was constructed by inserting themaize AdhIS first intron between the 35S promoter and the codingsequence of pBI221 (Jefferson et al. 1987). A map of pBII221 is providedas FIG. 2.

Plasmids were introduced into the embryogenic callus culture AB12 bymicroprojectile bombardment.

III. DNA Delivery Process

The embryogenic maize callus line AB12 was subcultured 7 to 12 daysprior to microprojectile bombardment. AB12 callus was prepared forbombardment as follows. Five clumps of callus, each approximately 50 mgin wet weight were arranged in a cross pattern in the center of asterile 60×15 mm petri plate (Falcon 1007). Plates were stored in aclosed container with moist paper towels, throughout the bombardmentprocess. Twelve plates were prepared.

Plasmids were coated onto M-10 tungsten particles (Biolistics) exactlyas described by Klein et al. (1988b) except that, (i) twice therecommended quantity of DNA was used, (ii) the DNA precipitation ontothe particles was performed at 0° C., and (iii) the tubes containing theDNA-coated tungsten particles were stored on ice throughout thebombardment process.

All of the tubes contained 25 μl of 50 mg/ml M-10 tungsten in water, 25μl of 2.5 M CaCl₂, and 10 μl of 100 mM spermidine along with a total of5 μl of 1 mg/ml plasmid DNA. When both plasmids were used, each waspresent in an amount of 2.5 μl. One tube contained only plasmid pBII221;and one tube contained T.E. buffer (see Table 2, below).

All tubes were incubated on ice for 10 min., the particles were pelletedby centrifugation in an Eppendorf centrifuge at room temperature for 5seconds, 25 μl of the supernatant was discarded. The tubes were storedon ice throughout the bombardment process. Each preparation was used forno more than 5 bombardments.

Macroprojectiles and stopping plates were obtained from Biolistics, Inc.(Ithaca, N.Y.). They were sterilized as described by the supplier. Themicroprojectile bombardment instrument was obtained from Biolistics,Inc.

The sample plate tray was placed 5 cm below the bottom of the stoppingplate tray of the microprojectile instrument, with the stopping plate inthe slot nearest to the barrel. Plates of callus tissue prepared asdescribed above were centered on the sample plate tray and the petridish lid removed. A 7×7 cm square rigid wire mesh with 3×3 mm mesh andmade of galvanized steel was placed over the open dish in order toretain the tissue during the bombardment. Tungsten/DNA preparations weresonicated as described by Biolistics, Inc. and 2.5 μl of the suspensionswere pipetted onto the top of the macroprojectiles for each bombardment.The instrument was operated as described by the manufacturer. Thebombardments which were performed are summarized on Table 2.

TABLE 2 2 × pBII221 prep To determine transient expression frequency 7 ×pHYGI1/pBII221 (1:1) As a potential positive treatment fortransformation 3 × T.E.^(a) Negative control treatment ^(a)10 mMTris·HCL, pH 8.0, 1 mM EDTA

The two plates of callus bombarded with pBII221 were transferred platefor plate to F medium (with no hygromycin) and the callus cultured at26° C. in the dark. after 2 days, this callus was then transferred platefor plate into 35×10 mm petri plates (Falcon 1008) containing 2 ml ofGUS assay buffer (1 mg/ml of5-bromo-4-chloro-3-indolyl-beta-D-glucuronide) (Research Organics), 100mM sodium phosphate pH 7.0, 5 mM each of potassium ferricyanide andpotassium ferrocyanide, 10 mM EDTA, and 0.06% Triton X-100. The plateswere incubated at 37° C. for 3 days after which the number of blue cellswas counted giving a total of 313 and 355 transient GUS-expressing cellsvisible in the two plates, suggesting that the DNA delivery process hadalso occurred with the other bombarded plates. The plates of tissue usedin the GUS assay were discarded after counting since the GUS assay isdestructive.

IV. Selection Process

Hygromycin B (Calbiochem) was incorporated into the medium prior topouring plates by addition of the appropriate volume offilter-sterilized 100 mg/ml hygromycin B dissolved in water, when themedium had cooled to 45° C.

Immediately after all samples had been bombarded, callus from all of theplates treated with pHYGI1/pBII221, and two of the T.E. plates wastransferred plate for plate onto F medium containing 15 mg/l hygromycinB, (ten pieces of callus per plate). These are referred to as round 1selection plates. Callus from the T.E. treated plate was transferred toF medium without hygromycin. This tissue was subcultured every 2-3 weeksonto nonselective medium and is referred to as unselected controlcallus.

After 14 days of selection, tissue appeared essentially identical onboth selective and nonselective media. All callus from seven plates ofthe pHYGI1/pBII221 and one T.E. treated plate were transferred fromround 1 selection plates to round 2 selection plates that contained 60mg/l hygromycin. The round 2 selection plates each contained ten 30 mgpieces of callus per plate, resulting in an expansion of the totalnumber of plates.

After 21 days on the round 2 selection plates, all of the material wastransferred to round 3 selection plates containing 60 mg/l hygromycin.After 79 days post-bombardment, the round 3 sets of selection plateswere checked for viable sectors of callus. One of the sectors wasproliferating from a background of necrotic tissue on plates treatedwith pHYGI1/pBII221. The sector was designated PH3 and was transferredto F medium without hygromycin.

After 19 days on F medium without hygromycin, PH3 was transferred toF-medium containing 60 mg/l hygromycin. PH3 was found to be capable ofsustained growth through multiple subcultures in the presence of 60 mg/lhygromycin.

V. Confirmation of Transformed Callus

To show that the PH3 callus had acquired the hygromycin resistance gene,genomic DNA was isolated from PH3 callus and unselected control callusin accord with Example V, and analyzed by Southern blotting. Theisolated DNA (10 μg) was digested with BamHI (NEB) and electrophoresedin a 0.8% w/v agarose gel at 15 V for 16 hrs in TAE buffer (40 mMTris-acetate, pH 7.6, 1 mM EDTA). The DNA was transferred to a Nytranmembrane (Schleicher and Schuell). Transfer, hybridization and washingconditions were carried out as per the manufacturer's recommendations.

A ³²P labelled probe was prepared by random primer labelling with anOligo Labelling Kit (Pharmacia) as per the supplier's instructions withα-³²P-dCTP (ICN Radio-chemicals). The template DNA used was the 1055 bpBamHI fragment of pHYGI1, which contains the entire HPT coding sequence.

Membranes were exposed to Kodak X-OMAT AR film in an X-OMATIC cassettewith intensifying screens. A band was observed for PH3 callus at theexpected position of 1.05 Kb, indicating that the HPT coding sequencewas present. No band was observed for control callus.

To demonstrate that the hygromycin gene is incorporated into highmolecular weight DNA, undigested DNA from PH3 callus and control calluswas electrophoresed, blotted and hybridized as described above.Undigested PH3 DNA only showed hybridization to the probe at a mobilityequal to uncut DNA. No hybridization was observed for DNA from controlcallus. These results demonstrate that the HPT coding sequence is notpresent in PH3 callus as intact pHYGI1 or as a small non-chromosomalplasmid. The results are consistent with incorporation of the hygromycingene into high molecular weight DNA.

VI. Plant Regeneration and Production of Seed

Portions of PH3 callus were transferred directly from plates containing60 mg/l hygromycin to RM5 medium which consists of MS basal salts(Murashige et al. 1962) supplemented with thiamine HCl 0.5 mg/l, 2,4-D0.75 mg/l, sucrose 50 g/l, asparagine 150 mg/l, and Gelrite 2.5 g/l(Kelco Inc., San Diego).

After 14 days on RM5 medium, the majority of PH3 and unselected controlcallus was transferred to R5 medium (RM5 medium, except that 2,4-D isomitted). The plates were cultured in the dark for 7 days at 26° C. andtransferred to a light regime of 14 hrs light and 10 hrs dark for 14days at 26° C. At this point, plantlets that had formed were transferredto one quart canning jars (Ball) containing 100 ml of R5 medium. Plantswere transferred from jars to vermiculite for 7 or 8 days beforetransplanting them into soil and growing them to maturity. A total of 45plants were produced from PH3 and a total of 10 plants were producedfrom control callus.

Controlled pollinations of mature PH3 plants were conducted by standardtechniques with inbred Zea mays lines MBS501 (Mike Brayton Seeds),FR4326 (Illinois Foundation Research) and the proprietary inbred lineLM1112. Seed was harvested 45 days post-pollination and allowed to dryfurther for 1-2 weeks.

VII. Analysis of the R1 Progeny

R1 plants were tested for the presence of the HPT and GUS gene sequencesby PCR analysis. Expression of the HPT gene was determined with anenzymatic assay for HPT activity. Table 3 summarizes the data. Thesedata demonstrate transmission and expression of the recombinant DNAthrough both male and female parents, and are consistent with Mendelianinheritance. The Southern blot evidence for integration of the HPTcoding sequence into high molecular weight DNA for the source callus,combined with the inheritance data, suggest that the foregoing DNAsequences are chromosomally integrated. The presence of GUS genesequences in the R1 progeny demonstrates cotransformation andinheritance of an unselected gene.

TABLE 3 Analysis of R1 Progeny of PH3 Transformants Enzyme PCR AssayPH3.4 Plant HPT Assay HPT GUS Parents: MBS501 × PH3.4 4.1 − − −4.2 + + + 4.3 + + + 4.4 − − − 4.5 + + + 4.6 + + + 4.7 + + + 4.8 − − −4.9 − − − Parents: LM1112 × PH3.18 18.1 − − − 18.2 + + + 18.3 − − − 18.4− − − 18.5 + + + 18.6 + + + 18.7 + + + 18.8 + + + 18.9 + + + 18.10 + + +Enzyme PCR Assay Plant HPT Assay HPT GUS Parents: PH3.2 × FR4326 2.1 − −− 2.2 − − − 2.3 + + + 2.4 − − − 2.5 + + + 2.6 − − − 2.7 + + + 2.8 + + ND2.9 + + ND Controls Parents: TE3.1 × Oh43 3.1 − − ND 3.2 − − ND 3.3 − −ND 3.4 − − ND 3.5 − − ND 3.6 − − ND 3.7 − − − 3.8 − − − 3.9 − − − 3.10 −− − 3.11 − ND ND 3.12 − ND ND 3.13 − ND ND 3.14 − ND ND 3.15 − ND ND3.16 − ND ND 3.17 − ND ND 3.18 − ND ND 3.19 − ND ND 3.20 − ND ND 3.21 −ND ND 3.22 − ND ND 3.23 − ND ND 3.24 − ND ND

The HPT enzyme assay was based on the following methods: S. K. Datta etal., Bio/Technology, 8, 736 (1990), M. Staebell et al., Anal. Biochem.,185, 319 (1990), and E. Cabanes-Bastos, Gene, 77, 169 (1989).

Root samples (0.2 g) were excised from 7-10 day old seedlings and werequick frozen in liquid nitrogen (N₂). Samples were ground with aluminaand 400 μl of EB (50 mM Tris-HCl, 10% v/v glycerol, 0.1 mM PMSF, pH 7.0)for 30-60 seconds using a disposable pestle and tube (Kontes), thencentrifuged in an Eppendorf microfuge for 10 min. The supernatant wastransferred to a Centricon 30 filter unit and desalted by centrifugingin a fixed angle rotor at 5000 rpm in a Beckman GPR centrifuge for 30min at 4° C. One ml of EB was used to wash each filter unit, spinning anadditional 5000 RPM for 60 min. The retentate was then recovered andstored at −70° C. Samples of transgenic and unselected control callustissue (0.2 g) were ground as described above, and the supernatents wereused as positive and negative controls.

Protein was quantified using the method of Bradford, Anal. Biochem., 72,248 (1976) with a BioRad kit. Protein concentrations in root extractsranged from 0.2-2 μg/μl.

The root extract (4 μg total protein) was added to a reaction mixturecontaining 20 mM Tris-maleate, 13 mM MgCl₂, 120 mM NH₄Cl, 0.5 mM DTT, 50μM ATP, 0.61 μg/μl hygromycin B, 25 μg/μl bovine serum albumin, and 12μCi gamma 32P-ATP. Reaction volume was 33 μl. The reaction mixture wasincubated for 30 min at 37° C.

One μl of the reaction mixture was spotted on a polyethyleneiminecellulose thin layer chromatography plate (Sigma Chem. Co.). Plates weredeveloped in 50 mM formic acid pH 5.4, air dried, and exposed to KodakXAR-5 film. The reaction product, hygromycin phosphate, migrates nearthe solvent front under these conditions.

To conduct the PCR assay, 0.1 g samples were taken from plant tissuesand frozen in liquid nitrogen. Samples were then ground with 120 gritcarborundum in 200 μl 0.1M Tris-HCl, 0.1M NaCl, 20 mM EDTA, 1% SarkosylpH 8.5) at 4° C. Following phenol/chloroform extraction and ethanol andisopropanol precipitations, samples were suspended in T.E. and analyzedby polymerase chain reaction (K. B. Mullis, U.S. Pat. No. 4,683,202).

PCR was carried out in 100 μl volumes in 50 mM KCl, 10 mM Tris-HCl pH8.4, 3 mM MgCl₂, 100 μg/ml gelatin, 0.25 μM each of the appropriateprimers, 0.2 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP,dTTP), 2.5 Units of Tag DNA polymerase (Cetus), and 10 μl of the DNApreparation. The mixture was overlaid with mineral oil, heated to 94° C.for 3 min, and amplified for 35 cycles of 55° C. for 1 min, 72° C. for 1min, 94° C. for 1 min. The mixture was then incubated at 50° C. for 2min and 72° C. for 5 min. 10 μl of the PCR product was electrophoresedin agarose gels and visualized by staining with ethidium bromide.

For analysis of the presence of the HPT gene, a PCR primer complementaryto the 35S promoter, and one complementary to the HPT coding sequencewere employed. Thus, in order to generate the appropriately sized PCRproduct, the HPT template DNA must contain contiguous 35S promoterregion, Adh1 intron, and 5′ protein coding sequence region.

For analysis of the presence of the GUS gene, PCR primers complementaryto sequences within the GUS protein coding region were employed. A 797bp PCR product is predicted if the template DNA contains an intactcoding region between the two primers.

Example II Fertile Transgenic Plants from Callus Line AB63S ContainingRecombinant DNA Encoding a Seed Storage Protein

The 10 kD-zein storage protein is produced in the endosperm of the maizekernel and is a representative member of a class of proteins referred toas “seed storage proteins.” The protein contains extremely high levelsof methionine (22.5%) and is encoded by the Zps10/(22) gene (M. S.Benner et al., Theor. Appl. Genet., 78, 761 (. 89)); a gene referred toas z10 herein. Thus, increased expression of the z10 gene can be used toincrease the methionine content of corn. Production of fertiletransgenic corn containing a chimeric z10 gene was accomplished by theprocedures of Example I with some minor modifications. Example II alsodemonstrates the introduction of recombinant DNA using a callus linedifferent from that used in Example I.

I. Tissue Culture Lines

The embryogenic maize callus line named AB63S used in this example wasproduced from immature embryos resulting from a cross between eliteinbred lines A188 and B73, by the initiation and maintenance proceduresdescribed in Example I. The callus line had been initiated approximately7 months prior to bombardment. Eleven weeks prior to bombardment thetissue was sieved by forcing it through a 1.9 mm screen and plated ontoN6 maintenance medium. Friable, embryogenic callus was selected from theresulting tissue after 1-2 weeks of growth, transferred to fresh mediumand allowed to grow for 1.5-3 weeks before sieving again. This cycle ofsieving and recovery of friable callus was carried out a total of threetimes prior to subjecting the tissue to bombardment.

II. Plasmids

The plasmids pHYGI1 and pBII221 described in Example I were used in thisExample. In addition, the plasmid pZ27Z10 was included in theDNA/tungsten preparations. The plasmid pZ27Z10 was constructed using thevector pUC118 (J. Vieira et al., Methods Enzymol., 153, 3 (1987), a 3.2kb circular plasmid, by standard recombinant DNA techniques. pZ27Z10contains the 5′ transcriptional regulatory region from a maize 27kD-zein gene (D. E. Geraghty, Ph.D. Thesis, University of Minnesota(1987)), referred to as z27 in this document), positioned immediatelyadjacent to the coding sequence and 3′ noncoding sequence from a maize10 kD-zein gene (Kirihara et al., Gene, 7, 359 (1988)), referred to asz10 in this document). The combination of the z27 regulatory sequenceand z10 coding and 3′ sequences is referred to as the chimeric z27-z10gene in this document. The poly A signal from the region of the CaMVgenome adjacent to the 35S promoter is positioned 3′ to the z10 genesequences in pZ27Z10. FIG. 7 depicts a map of this plasmid.

A sample of plasmid pZ27Z10 has been deposited in the American TypeCulture Collection, Rockville, Md., under the provisions of the BudapestTreaty under accession number ATCC 40938.

III. DNA Delivery Process

Tissue from the maize embryogenic callus line AB63S was subcultured 3weeks prior to microprojectile bombardment. The tissue was prepared forbombardment by sieving it through a 1.9 mm screen. The sieved tissue wasplated onto sterile 5.5 cm Whatman No. 1 filter disks as a thin lawn ofapproximately 500 mg of tissue per filter disk. A total of 6 disks oftissue were prepared. Prior to microprojectile bombardment the filterdisks containing the tissue were transferred to empty petri plates. Thetissue was dehydrated slightly by allowing the plates to stand uncoveredin a laminar flow hood for 20 minutes. To prevent further dehydration ofthe tissue, the plates were stored in a high humidity box until used forbombardment.

The DNA used in the bombardment consisted of a 1:1:1 mixture (by weight)of the plasmids pHYGI1, pZ27Z10 and pBII221. Precipitation of the DNAonto tungsten particles was carried out as described in Example I.

The bombardments were carried out as described in Example I except thatthe Biolistic™ PDS 1000 (DuPont) instrument was used, and no screen wasplaced over the tissue. The six filter disks with callus tissue weretreated as follows: two filters were bombarded two times each with theDNA/tungsten preparation (potential positive treatment 2×); threefilters were bombarded one time each with the DNA/tungsten preparation(potential positive treatment 1×); one filter was bombarded once with atungsten/water suspension using the same weight of tungsten used in theDNA/tungsten bombardments (negative control treatment).

IV. Selection of Transformed Callus

After bombardment, the filter disks with callus tissue from the 2×DNAtreatment were transferred to round 1 selection plates: F mediumcontaining 15 mg/l hygromycin. Two of the filters from the 1×DNAtreatment and the filter from the negative control treatment were alsotransferred to round 1 selection medium. The third filter from the 1×DNAtreatment was transferred to F medium containing no hygromycin, for useas unselected control callus. Since this unselected control callus waspotentially positive, it was only maintained for the first two rounds ofselection for comparative purposes; subsequently, non-bombarded AB63Scallus maintained on F medium without hygromycin was used as unselectedcontrol callus.

After five days the callus was transferred in small clumps (about 25 mg)from the filter disks onto fresh round 1 selection medium. The platingdensity was 10 callus clumps per plate. By this time the callus hadincreased approximately two-fold in weight. The unselected controlcallus was transferred in the same manner to F medium withouthygromycin. At nineteen days post-bombardment, all of the tissue wastransferred from round 1 selection medium to round 2 selection medium (Fmedium containing 60 mg/l hygromycin). The plating density was 14 callusclumps per plate at this transfer. The callus had increasedapproximately three-fold in volume during the 14 days since the previoustransfer. After 23 days, all of the callus was transferred from round 2selection medium to round 3 selection medium containing 60 mg/lhygromycin.

At 59 days post-bombardment, five live sectors were observedproliferating among the surrounding necrotic callus clumps. These fivesectors were thought to have been derived from a single callus clumpfrom the round 2 selection, since they appeared adjacent to one anotheron consecutive plates from the previous transfer. These sectors arosefrom a 2×DNA treatment and were designated as callus line Met1.

The callus line Met1 was transferred to F medium containing 60 mg/lhygromycin and to F medium without hygromycin. After 22 days ofincubation, there was no visible difference in growth or appearance ofthe tissue on the two types of media. The callus line Met1 grew rapidlyand appeared to be uninhibited by the presence of the hygromycin in themedium. The Met1 callus appeared highly friable and embryogenic oneither medium, and was visually indistinguishable from control AB63Scallus grown on F medium without hygromycin.

V. Confirmation of Transformed Callus

An inhibition study was performed using Met1 callus and control AB63Scallus. Prior to initiating the inhibition study, Met1 callus was grownfor 21 days on F medium without hygromycin. Callus of Met1 and controlAB63S callus was transferred onto plates of F medium containing 0, 15,60, 100 and 200 mg/l hygromycin. Three plates of each callus line wereprepared for each concentration of hygromycin; each plate contained 5 or6 pieces of callus at approximately 50 mg per piece (total callus perplate was approximately 300 mg).

After 28 days of incubation, the weight of callus on each plate wasmeasured. A measure of percent growth inhibition was determined bydividing the average weight of callus obtained at each hygromycinconcentration by the average weight of callus obtained at 0 mg/lhygromycin. The results showed that the growth of Met1 callus wascompletely uninhibited at hygromycin concentrations of 15, 60 and 100mg/l. The growth of Met1 callus was approximately 20% inhibited onmedium containing 200 mg/l hygromycin. In contrast, the growth of AB63Scallus was inhibited at all concentrations of hygromycin; at 200 mg/lhygromycin, AB63S callus was 90% inhibited in growth. These resultsconfirmed that the callus line Met1 exhibited resistance to the presenceof hygromycin in the growth medium.

Southern blot analysis was carried out to verify the presence of thehygromycin resistance gene in Met1 callus DNA and to determine whetherthe callus had also acquired the chimeric z27-z10 gene. For detection ofthe HPT coding sequence, DNA samples isolated from Met1 callus and fromcontrol callus were digested with the restriction enzymes BamHI, HindIIIand BstEII. The digested DNA samples were subjected to electrophoresison a 0.8% agarose gel and blotted to a Nytran membrane as described inExample I. Visual inspection of the ethidium bromidestained gel prior toblotting indicated that the BamHI digestions appeared to be complete,whereas neither HindIII nor BstEII cleaved the DNA samples to asignificant degree. The blot was probed with a biotin-labeled 1.05 kbBamHI fragment from the HPT coding sequence. The conditions used forhybridization, washing and detection were as suggested in a BRLPhotogene™ kit used in this experiment.

After hybridization, labeled bands were observed in the lanes containingBamHI digested Met1 DNA at the expected size of 1.05 kb as well as atsizes of approximately 2.7, 4.5 and 6.5 kb. This result showed that theHPT coding sequence was present in DNA from the callus line Met1. Theadditional bands at 2.7, 4.5 and 6.5 kb indicated that either therestriction enzyme digestion was incomplete, or that multiple rearrangedcopies of the HPT sequence were present. For the HindIII and BstEIIdigests, hybridization signals were visible in both lanes at theposition of undigested DNA. This result indicated that the HPT codingsequence was integrated into high molecular-weight DNA in the Met1genome. No hybridization signals were observed in any of the lanescontaining digestions of DNA from control callus.

For detection of the chimeric z27-z10 gene, Southern blot analysis wascarried out using DNA samples isolated from Met1 callus and controlcallus. The DNA samples were digested with the restriction enzymes BamHIand EcoRI. A BamHI digestion liberates a 2.76 kb fragment containing thez10 coding sequence and 3′ noncoding sequence from the pZ27Z10construction used in this transformation. There is a single EcoRI sitein the 7.26 kb pZ27Z10 plasmid. The blot prepared was hybridized with abiotin-labeled probe containing the entire z10 coding sequence, usingthe conditions described in a Photogene kit (BRL).

For the BamHI digestions, endogenous z10 sequences gave hybridizationsignals at 9 kb and at approximately 15 kb in the lanes containing bothMet1 and control callus DNAs. An additional band was observed atapproximately 3.5 kb in the Met1 sample but not in the control DNAsamples. No strong hybridization signal was observed at the expected2.76 kb size in Met1 DNA. The lack of a labeled band of 2.76 kb in theBamHI digestion of Met1 DNA indicated either incomplete digestion of theDNA, or rearrangement of the introduced DNA.

For the EcoRI digestions, endogenous z10 sequences gave hybridizationsignals at approximately 12 kb and 16 kb in both Met1 and control callusDNAs. An additional band was observed at approximately 14 kb in the Met1sample but not in the control callus DNA samples. These resultsindicated that novel z10 sequences were present in DNA from Met1 callusthat were absent from control callus DNA samples.

These results were confirmed by a second Southern blot analysis in whichMet1 and control callus DNAs were digested with BamHI, NcoI and NsiI.Also included were samples of undigested DNA from Met1 and controlcallus. The filter was probed with a ³²P-labeled z10 coding sequenceprobe. In all of the digestions, novel z10 hybridization signals wereobserved in Met1 callus DNA that were absent in the negative controlcallus DNA samples. These results confirmed that novel z10 codingsequences were present in the Met1 callus. In the lanes containingundigested DNA, hybridization signals were observed at the position ofundigested DNA from both Met1 and control callus. These resultsindicated that the introduced z10 sequences were integrated intochromosomal DNA in Met1 callus.

VI. Plant Regeneration and Production of Transgenic Seed

Callus from the line Met1 and from the control line AB63S was placed onRM5 medium as described in Example I. After 14 days of incubation at 26°C. in the dark, the callus was transferred to R5 medium as in Example I.The callus was incubated at 26° C. in the dark for 7 days, and thentransferred to the light under the conditions described in Example I.After 14 days in the light, plantlets were transferred to Magenta boxes(Sigma Chemical Co.) containing 100 ml of R5 medium. After 7-14 days onthis medium, plantlets were transferred to vermiculite for 7 days andthen to soil where they were grown to maturity. A total of 49 plants(designated Met1-1 through Met1-49) were regenerated from Met1 callus,and a total of 25 plants were regenerated from control AB63S callus.

PCR analysis was carried out on samples of DNA prepared from leaf tissueto verify that the plants regenerated from Met1 callus had retained theintroduced DNA. For this purpose, a set of six oligodeoxyribonucleotideswere used. The sequences of these oligonucleotides, their orientationsand relative positions within the chimeric z27-z10 gene constructionshown in FIG. 8 are summarized in Table 4 below.

TABLE 4 I. OLIGONUCLEOTIDE NAMES/POSITIONS IN CHIMERIC Z27-Z10 GENE: 1.Z27-5′ nt 132-156 2. z27-MID nt 628-651 3. Z27-3′ nt 1081-1104 4. Z10-5′nt 1182-1206 5. Z10-MID nt 1420-1444 6. Z10-3′ nt 1548-1571 II.AMPLIFIED FRAGMENT SIZES FROM OLIGONUCLEOTIDE PAIRS: A. ENDOGENOUS ORCHIMERIC GENE PAIRS: (1) + (3)  972 BP (2) + (3)  476 BP (4) + (6)  389BP (4) + (5)  262 BP B. CHIMERIC GENE-SPECIFIC PAIRS: (1) + (6) 1439 BP(1) + (5) 1312 BP (2) + (6)  943 BP (2) + (5)  816 BP

The set of oligonucleotides (or primers) was designed such that the useof a pair of oligonucleotides consisting of one z27 oligonucleotide(z275′ or z27mid) and one z10 oligonucleotide (z10mid or z103′) wouldresult in amplification of fragments from the chimeric z27-z10 geneonly, and not from endogenous maize z27 or z10 genes. The appearance ofamplified fragments of the expected sizes in PCR reactions using thesez27-z10 -specific oligonucleotide pairs were diagnostic for the presenceof the chimeric z27-z10 gene in a particular callus or plant DNA sample.

The use of a pair of z27 oligonucleotides (z275, or z27mid with z273′)or a pair of z10 oligonucleotides (z105′ with z10mid or z103′) resultedin amplification of fragments representing z27 regulatory sequences orz10 coding sequences, respectively, when used in PCR reactions carriedout with control AB63S callus DNA, Met1 callus DNA or pZ27Z10 plasmidDNA. These reactions served as positive controls for amplification ofendogenous maize gene sequences from a particular callus or plant DNAsample.

Leaf DNA was prepared from two Met1 R0 plants (designated Met1-1 andMet1-2) and from one control AB63S R0 plant as described hereinbelow.PCR reactions were carried out with these DNA samples using theoligonucleotide pairs specific for the HPT coding sequence, the chimericz27-z10 gene, the z27 regulatory sequence and the z10 coding sequence.Gel analysis of the PCR reaction products showed that the expected sizeamplification products were obtained in all reactions using Met1-1 DNAand Met1-2 DNA. Control AB63S DNA was negative for the HPT codingsequence and for the chimeric z27-z10 gene, but positive for theendogenous z27 regulatory sequence and z10 coding sequence. Theseresults demonstrated that the Met1 R0 plants examined had retained theintroduced HPT DNA and the chimeric z27-z10 DNA.

As corroborative proof that the diagnostic z27-z10 PCR reaction productscontained both z27 regulatory sequences and z10 coding sequences,Southern blot analysis was carried out on the PCR reaction products.Southern blot filters were prepared from two agarose gels loaded withidentical samples. The samples contained the PCR reaction productsresulting from amplification of z27 regulatory sequences, z10 codingsequences, and chimeric z27-z10 gene sequences described above. One blotwas hybridized with a z10 coding sequence probe and the other blot washybridized with a z27 regulatory sequence probe. The results showed thatthe z10 coding sequence probe hybridized to the amplified fragments fromthe z10 coding sequence PCR reactions and the chimeric z27-z10 gene PCRreactions, but not to the amplified fragments from the z27 regulatorysequence PCR reactions. Analogously, the z27 probe hybridized to theamplified fragments from the z27 regulatory sequence PCR reactions andthe chimeric z27-z10 gene PCR reactions but not to the amplifiedfragments from the z10 coding sequence PCR reactions. These resultsdemonstrated that the fragments amplified in the PCR reactions containedthe expected gene sequences and that the diagnostic PCR amplificationproduct for the chimeric z27-z10 gene contained both z27 regulatorysequences and z10 coding sequences.

Controlled pollinations of mature Met1 R0 plants were carried out withinbred maize lines A188, B73, A654 and H99. In addition, several self-and sib-pollinations were carried out. A total of 130 pollinations werecarried out, using Met1 R0 plants as both male pollen donors and asfemale pollen recipients. Mature seed was harvested at 45 dayspost-pollination and allowed to dry further for 1-2 weeks.

VII. Analysis of the R1 Progeny

The presence of the HPT sequence and the chimeric z27-z10 gene wereevaluated by PCR analysis of DNA from R1 plant tissues, in three sets ofR1 progeny.

The first set of R1 progeny analyzed consisted of four immaturetassel-seeds from R0 plant Met1-7. These tassel-seeds were obtainedthrough open pollination of silks developing from the tassel of Met1-7.Approximately 18 days after pollination, the tassel-seeds were removedfrom the plant and surface-sterilized. The endosperm from each seed wasexcised and frozen on dry ice and stored at −70° C. until used for DNAisolation.

The embryo from each seed was transferred to R5 medium and allowed togerminate. After 10 days, the seedlings were transferred to vermiculitefor 7 days and then to soil, where they were allowed to grow tomaturity. PCR analysis was carried out on DNA isolated from eachendosperm using oligonucleotide sets specific for the HPT codingsequence, the chimeric z27-z10 gene and the maize Adh-1 gene. Three ofthe four endosperm DNA samples were found to be positive for all of theabove gene sequences. The fourth endosperm DNA sample was negative forthe HPT coding sequence and the chimeric z27-z10 gene sequence butpositive for the endogenous Adh-1 sequence. These results showed thatboth the HPT sequence and the chimeric z27-z10 gene had been transmittedto R1 progeny of Met1-7.

A similar analysis was carried out with 24 seeds from the crossA654×Met1-1. At 24 days after pollination, the seeds were removed fromthe ear and surface-sterilized. Endosperms and embryos were isolated andtreated as described above. PCR analysis of the endosperm DNA samplesshowed that 9 of the 24 progeny had received both the HPT sequences andthe chimeric z27-z10 gene. The remaining 15 progeny carried neither ofthe introduced gene sequences. The third set of R1 progeny analyzedconsisted of 28 R1 plants derived from self-pollination of R0 plantMet1-6. DNA samples were prepared from leaf tissue of 28 two-week oldseedlings.

PCR analysis of the leaf DNA samples showed that 24 of the R1 progenycarried both the HPT sequences and the chimeric z27-z10 gene. Theremaining 4 R1 progeny carried neither of the introduced gene sequences.The results of the analyses described above are summarized in Table 5and demonstrate that a significant proportion of the R1 progeny of Met1plants inherited the introduced DNA.

TABLE 5 Inheritance of the Introduced HPT Sequences and the Chimeric Z10Gene in Progeny from Crosses of Met1 Plants Outcross Self-Pollination(A654 × Met1-1) (Met1-6) Genotype No. Progeny No. Progeny HPT+/z27z10+9/24 24/28  HPT+/z27z10− 0/24 0/28 HPT−/z27z10+ 0/24 0/28 HPT−/z27z10−15/24  4/28

The ratio of z27z10+/HPT+ plants to z27z10−/HPT− plants from theoutcross population is consistent with a 1:1 segregation ratio of asingle locus. In addition, the ratio of z27z10+/HPT+ plants toz27z10−/HPT− plants from the self-pollinated population is consistentwith a 3:1 segregation ratio of a single locus. The data also indicatelinkage of the introduced HPT and z27-z10 sequences.

Example III Fertile Transgenic Plants from Callus Line AB12 ContainingRecombinant DNA Encoding an Insecticidal Protein

The proteins encoded by the various Bacillus thuringiensis genes havebeen shown to be useful in a number of pesticidal applications.Production of transgenic maize plants with specific heterologous Btgenes may improve the resistance of the plants to specific pests.Fertile transgenic plants containing a recombinant Bt gene were preparedaccording to the procedure of Example I, with several minormodifications, as indicated below.

The plasmid pHYGI1 described in Example I was used, as well as pMS533,which is a plasmid that contains the insecticidal Bacillus thuringiensisendotoxin (BT) gene fused in frame with the neomycin phosphotransferase(NPTII) gene. At a position 5′ from the fusion gene are located segmentsof DNA from the CaMV and nopaline synthase promoters. At a position 3′from the fusion gene are segments of DNA derived from the tomatoprotease inhibitor I gene and the poly A region of the nopaline synthasegene.

AB12 callus was bombarded as described in Example I except that the DNAused in the tungsten/DNA preparations contained plasmids pHYGI1 andpMS533. One tube contained only 5 μl TE (10 mM Tris-HCl pH 8.0, 1 mMEDTA).

The following bombardments were done: 11×pHYGI1/pMS533 (potentialpositive treatment) and 3×TE prep (control treatment).

After bombardment, the callus from the pHYGI1/pMS533 treatments wasplaced onto round 1 selection plates, F medium containing 15 mg/lhygromycin as ten pieces per plate. The same was done for two of theplates bombarded with the TE preparation (selected control callus). Oneplate of callus bombarded with the TE preparation was placed onto Fmedium with no hygromycin; this callus was maintained throughout theexperiment as a source of control tissue (unselected control callus).

After 18 days, the callus was transferred from round 1 selection platesto round 2 selection plates containing 60 mg/l hygromycin as ten 30 mgpieces per plate. After 21 days of selection on round 2 selectionplates, the callus appeared completely inhibited. All of the callus wastransferred from round 2 selection plates to round 3 selection platescontaining 60 mg/l hygromycin.

After 42 days on round three selection plates, six sectors were observedproliferating from the surrounding necrotic tissue, all were frompHYGI1/pMS533-treated material. The callus lines were transferred to Fmedium.

After 24 days, one callus line, designated CB2, had grown substantially.Portions of each callus line were then transferred to (i) F mediumcontaining 15 mg/l hygromycin, or (ii) F medium containing 60 mg/lhygromycin. Control callus was plated on F medium with 15 mg/lhygromycin.

Only one of the six callus lines, CB2, was capable of sustained growththrough multiple subcultures in the presence of 60 mg/l hygromycin. DNAwas isolated from portions of this callus and the presence of the Btgene was confirmed by Southern blot analysis.

DNA was digested with the restriction enzymes BamHI and XhoI incombination. BamHI cuts at the 5′ end of the Bt coding sequence and XhoIcuts at the 3′ end of the coding region. A ³²P labeled probe wasprepared from the 1.8 kb BamHI/XhoI restriction fragment of pMS533. Thepredicted 1.8 kb band was observed after hybridization andautoradiography. No bands were observed in the DNA from theuntransformed callus.

Plants were regenerated from CB2 and controlled pollinations wereperformed to yield the R1 generation, as described hereinabove. R1plants were tested for the presence of the HPT and Bt gene sequences byPCR analysis, and for HPT gene expression by enzyme assay as describedin Example I.

PCR analysis was performed on DNA isolated from root tissue of R1seedlings by the methods described in Example I. To test that each DNAsample was competent for PCR, PCR analysis was carried out using primersspecific for the native maize 10 kD zein gene. The primers used for PCRanalysis of the Bt gene were one complementary to the 35S promoter andone complementary to the Bt coding sequence. Primers used for HPT PCRanalysis were the same as those described in Example I. The results ofthis analysis are presented in Table 6. The results show transmission ofthe introduced HPT and Bt genes through both the male and female parentsand the observed frequencies are consistent with Mendelian inheritance.The results are consistent with the insertion of the HPT and Bt genesinto chromosomal DNA. It was also found that the HPT and Bt genes show alinked inheritance, indicating that the two genes are inserted cosetogether on the same chromosome.

TABLE 6 Analysis of R1 Progeny of CB2 A. Transgenic Control CB2.8 ×FR4326 AB12.7 × FR4326 Analysis Analysis 10Kd HPT BT HPT 10Kd HPT BT HPTPlant PCR PCR PCR ENZ. PLANT PCR PCR PCR ENZ. 1 + + + + 1 + − − −2 + + + + 2 + − − − 3 + − − − 4 + + + + 5 + + + + 6 + − − − 7 + + + +8 + − − − 9 + + + + 10  + − − − B. Transgenic Control CB2.11 × LB101AB12.4 × LB101 Assays Assays 10Kd HPT BT HPT 10Kd HPT BT HPT Plant PCRPCR PCR ENZ. PLANT PCR PCR PCR ENZ. 1 + − − − 1 + − − − 2 + + + + 2 + −− − 3 + − − − 4 + + + + 5 + − − − 6 + + + + 7 + − − − 8 + − − − 9 + − −− 10  + − − + C. Transgenic Control CB2.7 × LM1112 AB12.5 × LM1112Assays Assays 10Kd HPT BT 10Kd HPT BT Plant PCR PCR PCR PLANT PCR PCRPCR 1 + − − 1 + − − 2 + + + 2 + − − 3 + − − 4 + − − 5 + + + 6 + + +7 + + + 8 + + + 9 + − − 10  + − − D. Transgenic Control FR4326 × CB2.9FR4326 × AB12.1 Assays Assays 10Kd HPT BT 10Kd HPT BT Plant PCR PCR PCRPLANT PCR PCR PCR 1 + − − 1 + − − 2 + + + 2 + − − 3 + + + 4 + + +5 + + + 6 + − − 7 + + + 8 + + + 9 + − − 10  + − − E. Transgenic ControlA188 × CB2.1 A188 × AB12.2 Assays Assays 10Kd HPT BT 10Kd HPT BT PlantPCR PCR PCR PLANT PCR PCR PCR 1 + + + 1 + − − 2 + + + 2 + − − 3 + + +4 + − − 5 + + + 6 + − − 7 + + + 8 + + + 9 + − − 10  + − − F. TransgenicControl LM1112 × CB2.2 LM112 × AB12.6 Assays Assays 10Kd HPT BT 10Kd HPTBT Plant PCR PCR PCR PLANT PCR PCR PCR 1 + − − 1 + − − 2 + − − 2 + − −3 + − − 4 + − − 5 + − − 6 + − − 7 + − − 8 + +

Example IV Fertile Transgenic Plants from Callus Line AB12 TransmittingRecombinant DNA through Two Complete Sexual Cycles

I. Plant Lines and Tissue Culture

The callus line AB12 was used, and is described in Example I.

II. Plasmids

The plasmids pBII221 and pHYGI1 described in Example I were used.

III. DNA Delivery Process

Callus was bombarded as in Example I, except that the DNA used in thetungsten/DNA preparations differed. All of the tubes contained 25 μl 50mg/ml M-10 tungsten in water, 25 μl 2.5 M CaCl₂, and 10 μl 100 mMspermidine along with a total of 5 μl 1 mg/ml total plasmid content. Onetube contained only plasmid pBII221; two tubes contained only plasmidpHYGI1; and one tube contained no plasmid but 5 μl TE buffer.

The following bombardments were done: 2×pBII221 prep (for transientexpression); 7×pHYGI1 prep (potential positive treatment); and 3×TE prep(negative control treatment).

After all the bombardments were performed, the callus from the pBII221treatment was transferred plate for plate to F medium as five 50 mgpieces. After 2 days, the callus was placed into GUS assay buffer as perExample I. Numbers of transiently expressing cells were counted andfound to be 686 and 845 GUS positive cells, suggesting that the particledelivery process had occurred in the other bombarded plates.

IV. Selection of Transformed Callus

After bombardment, the callus from the pHYGI1 treatments was placed ontoround 1 selection plates, F medium containing 15 mg/l hygromycin, as ten25 mg pieces per plate. The same was done for two of the platesbombarded with the TE preparation (selected control callus). One plateof callus bombarded with the TE preparation was placed onto F mediumwith no hygromycin; this callus was maintained throughout the experimentas a source of control tissue (unselected control callus).

After 13 days, the callus on round 1 selection plates wasindistinguishable from unselected control callus. All of the callus wastransferred from round 1 selection plates to round 2 selection platescontaining 60 mg/l hygromycin. An approximate five-fold expansion of thenumbers of plates occurred.

The callus on round 2 selection plates had increased substantially inweight after 23 days, but at this time appeared close to necrotic. Allof the callus was transferred from round 2 selection plates to round 3selection plates containing 60 mg/l hygromycin.

At 58 days post-bombardment, three live sectors were observedproliferating from the surrounding dead tissue. All three lines werefrom pHYGI1 treatments and were designated 24C, 56A, and 55A.

After 15 days on maintenance medium, growth of the lines was observed.The line 24C grew well whereas lines 55A and 56A grew more slowly. Allthree lines were transferred to F medium containing 60 mg/l hygromycin.Unselected control callus from maintenance medium was also plated to Fmedium containing 60 mg/l hygromycin.

After 19 days on 60 mg/l hygromycin, the growth of line 24C appeared tobe entirely uninhibited, while the control showed approximately 80% ofthe weight gain of 24C. The line 56A was completely necrotic, and theline 55A was very close to necrotic. The lines 24C and 55A weretransferred again to F medium containing 60 mg/l hygromycin, as was thecontrol tissue.

After 23 days on 60 mg/l hygromycin, the line 24C again appearedentirely uninhibited. The line 55A was completely dead, as was thenegative control callus on its second exposure to F medium having 60mg/l hygromycin.

V. Confirmation of Transformed Callus

A Southern blot was prepared from BamHI-digested DNA from the line 24Cand probed with the HPT probe described in Example I. As shown in FIG.6, a band was observed for the line 24C at the expected size of 1.05 kbshowing that the line 24C contained the HPT coding sequence. No band wasobserved for DNA from control tissue. The name of the callus line 24Cwas changed to PH2.

To demonstrate that the hygromycin gene is incorporated into highmolecular weight DNA, DNA isolated from PH2 callus and control calluswas treated with (i) no restriction enzyme, (ii) BamHI, as describedpreviously, or, (iii) PstI, which cuts the plasmid pHYGI1 only oncewithin the HPT coding sequence. Samples were blotted and probed with theHPT coding sequence as described previously.

Undigested PH2 DNA only showed hybridization to the probe at theposition of uncut DNA, demonstrating that the hygromycin gene isincorporated into high molecular weight DNA. The expected 1.05 kb bandfor PH2 DNA digested with BamHI was observed, as had been shownpreviously. For PH2 DNA digested with PstI, a 5.9 kb band would beexpected if the hygromycin gene was present on an intact pHYGI1 plasmid.Two or more bands of variable size (size dependent on the position offlanking PstI sites within the host DNA) would be expected if the genewas integrated into genomic DNA. Two bands were observed withapproximate molecular sizes of 6.0 and 3.0 kb. These data are consistentwith incorporation of the hygromycin gene into high molecular weightDNA. No hybridization was observed for DNA from control callus in any ofthe above treatments.

These results demonstrate that the HPT coding sequence is not present inPH2 callus as intact pHYGI1 or as a small non-chromosomal plasmid. Theyare consistent with incorporation of the hygromycin gene into highmolecular weight DNA. Further Southern blot analyses demonstrated thatthe HPT coding sequence is contiguous with the 35S promoter sequence.

VI. Plant Regeneration and Production of Seed

The line PH2, along with unselected control callus, was placed onto RM5medium to regenerate plants as in Example I. After 16 days, the calluswas transferred to R5 medium as in Example I. After 25 d on R5 medium,plantlets were transferred to R5 medium and grown up for 20 days. Atthis point, plantlets were transferred to vermiculite for one week andthen transplanted into soil where they were grown to sexual maturity.Controlled pollinations were then performed with the inbred line B73.

VII. Analysis of R1 Progeny

A. PH2 as Pollen Donor

Ten R1 progeny plants from a B73×PH 2.6 cross were assayed for HPTexpression with both root elongation and etiolated leaf assays. Onepositive plant was identified. The presence of the HPT gene in thepositive plant was confirmed by Southern blot analysis employing the HPTprobe as described in Example I.

To conduct the root elongation bioassay, seed was sterilized in a 1:1dilution of commercial bleach in water plus 0.1% Alconox for 20 min. in125 ml Erlenmeyer flasks and rinsed 3 times in sterile water. The seedswere imbibed overnight in sterile water containing 50 mg/ml Captan byshaking at 150 rpm.

After imbibition, the solution was decanted from the flasks and the seedtransferred to flow boxes (Flow Laboratories) containing 3 sheets of H₂Osaturated germination paper. A fourth sheet of water saturatedgermination paper was placed on top of the seed. Seed was allowed togerminate 4 days.

After the seed had germinated, approximately 1 cm of the primary roottip was excised from each seedling and plated on medium containing MSsalts, 20 g/l sucrose, 50 mg/l hygromycin, 0.25% Gelrite, and incubatedin the dark at 26° C. for 4 days.

Roots were evaluated for the presence or absence of abundant root hairsand root branches. Roots were classified as transgenic (hygromycinresistant) if they had root hairs and root branches, and untransformed(hygromycin sensitive) if they had limited numbers of branches.

After the root tips were excised, the seedlings of one PH2 ear and onecontrol ear were transferred to moist vermiculite and grown in the darkfor 5 days. At this point, to conduct an etiolated leaf bioassay, 1 mmsections were cut from the tip of the coleoptile, surface sterilized 10seconds, and plated on MS basal salts, 20 g/l sucrose, 2.5 g/l Gelritewith either 0 (control) or 100 mg/l hygromycin and incubated in the darkat 26° C. for 18 hrs. Each plate contained duplicate sections of eachshoot. The plates were then incubated in a light regimen of 14 hrs light10 hrs dark at 26° C. for 48 hrs, and rated on a scale of from 0 (allbrown) to 6 (all green) for the percent of green color in the leaftissue. Shoots were classified as untransformed (hygromycin sensitive)if they had a rating of zero and classified as transformed (hygromycinresistant) if they had a rating of 3 or greater.

B. PH2 as Pollen Recipient

80 R1 progeny plants from a PH2 plant designated PH 2.23×B73 cross wereassayed for HPT expression by root elongation assay and 26 testedpositive. The expression of the HPT gene was further confirmed for all26 by direct exposure of seedlings to hygromycin-containing medium inFlow boxes. Two of the resistant plants were analyzed by PCR, and thepresence of the HPT sequence was confirmed.

VIII. Analysis of R2 Progeny

The transgenic R1 plant which had PH2 as the pollen parent was grown tomaturity and used to pollinate an FR4326 plant.

R2 seeds from this cross were germinated and tested for the presence ofHPT sequences by PCR. Expression of the HPT gene was determined with theHPT enzymatic assay described in Example I.

Three of the 19 progeny tested contained HPT gene sequences and alsoexpressed the HPT enzyme activity. The remainder neither contained HPTsequences nor showed enzyme activity. This result demonstrates pollentransmission and expression of the genetically engineered DNA through 2successive generations, indicating that the gene is stable.

Example V

The friable, embryogenic maize callus culture AB12 described in ExampleI was used.

The plasmids pCHN1-1 and pLUC-1 were constructed in the vector pBS+(Stratagene, Inc., San Diego, Calif.), a 3.2 Kb circular plasmid, usingstandard recombinant DNA techniques. pCHN1-1 contains the hygromycin Bphosphotransferase (HPT) coding sequence from E. coli (Gritz et al.1983) flanked at the 3′ end by the nopaline synthase (nos)polyadenylation sequence of Agrobacterium tumefaciens (Chilton andBarnes 1983). Expression is driven by the cauliflower mosaic virus(CaMV) 35S promoter (Guilley et al. 1982), located upstream from the HPTcoding sequence. The plasmids pBII221 and pHYGI1 were also used in thisexample.

pLUC-1 contains the firefly luciferase coding sequence (DeWet et al.1987) flanked at the 5′ end by the CaMV 35S promoter and at the 3′ endby the nos polyadenylation sequence. This plasmid was used solely asnegative control DNA. Plasmids were introduced into AB12 bymicroprojectile bombardment. Twenty-six plates were prepared asdescribed in Example I. One tube contained only plasmid pBII221; twotubes contained both plasmids pHYGI1 and pBII221; two tubes containedboth plasmids pCHN1-1 and pBII221; and one tube contained only plasmidpLUC-1. The bombardments which were performed are summarized on Table 7.

TABLE 7  2 × pBII221 prep To determine transient expression frequency 10× pHYGI1/pBII221 As a potential positive treatment for transformation 10× pCHN1-1/pBII221 As a potential positive treatment for transformation 4 × pLUC-1 Negative control treatment

The two plates of callus bombarded with pBII221 were assayed fortransient GUS expression. The number of blue cells was counted, giving291 and 477 transient GUS expressing cells in the two plates, suggestingthat the DNA delivery process had also occurred with the other bombardedplates.

Immediately after all samples had been bombarded, callus from all of theplates treated with pHYGI1/pBII221, pCHN1-1/pBII221 and three of theplates treated with pLUC-1 were transferred plate for plate onto round 1selection plates, F medium containing 15 mg/l hygromycin B, (five piecesof callus per plate). Callus from the fourth plate treated with pLUC-1was transferred to F medium without hygromycin. This tissue wassubcultured every 2-3 weeks onto nonselective medium and is referred toas unselected control callus.

After two weeks of selection, tissue appeared essentially identical onboth selective and nonselective media. All callus from eight plates fromeach of the pHYGI1/pBII221 and pCHN1-1/pBII221 treatments and two platesof the control callus on selective media were transferred from round 1selection plates to round 2 selection plates that contained 60 mg/lhygromycin. The round 2 selection plates each contained ten 30 mg piecesof callus per plate, resulting in an expansion of the total number ofplates.

The remaining tissue on selective media, two plates each ofpHYGI1/pBII221 and pCHN1-1/pBII221 treated tissue and one of controlcallus, were placed in GUS assay buffer at 37° C. to determine whetherblue clusters of cells were observable at two weeks post-bombardment.After 6 days in assay buffer, this tissue was scored for GUS expression.The results are summarized on Table 8.

TABLE 8 Treatment Replicate Observations pLUC-1 No blue cellspHYGI1/pBII221 Plate 1 11 single cells 1 four-cell cluster Plate 2 5single cells pCHN1-1/pBII221 Plate 1 1 single cell 2 two-cell clustersPlate 2 5 single cells 1 two-cell cluster 2 clusters of 8-10 cells

After 21 days on the round 2 selection plates, all viable portions ofthe material were transferred to round 3 selection plates containing 60mg/l hygromycin. The round 2 selection plates, containing only tissuethat was apparently dead, were reserved. Both round 3 selection plateswere observed periodically for viable proliferating sectors.

After 35 days on round 3 selection plates, both the round 2 and round 3sets of selection plates were checked for viable sectors of callus. Twosuch sectors were observed proliferating from a background of deadtissue on plates treated with pHYGI1/pBII221. The first sector named3AA, was from the round 3 group of plates and the second sector namedPH1 was from the round 2 group of plates. Both lines were thentransferred to F medium without hygromycin.

After 19 days on F medium without hygromycin, the line 3AA grew verylittle whereas the line PH1 grew rapidly. Both were transferred again toF medium for 9 days. The lines 3AA and PH1 were then transferred to Fmedium containing 15 mg/l hygromycin for 14 days. At this point, line3AA was observed to be of very poor quality and slow growing. The linePH1, however, grew rapidly on F medium with 15 mg/l hygromycin; the linewas then subcultured to F medium without hygromycin.

After 10 days on F medium, an inhibition study of the line PH1 wasinitiated. Callus of PHi was transferred onto F medium containing 1, 10,30, 100, and 250 mg/l hygromycin B. Five plates of callus were preparedfor each concentration and each plate contained ten approximately 50 mgpieces of callus. One plate of unselected control tissue was preparedfor each concentration of hygromycin.

It was found that the line PH1 was capable of sustained growth over 9subcultures on 0, 10, 30, 100, and 250 mg/l hygromycin.

Additional sectors were recovered at various time points from the round2 and 3 selection plates. None of these were able to grow in thepresence of hygromycin for multiple rounds, i.e., two or threesubcultures.

To show that the PH1 callus had acquired the hygromycin resistance gene,a Southern blot of PH1 callus was prepared by isolating DNA from PH1 andunselected control calli by freezing 2 g of callus in liquid nitrogenand grinding it to a fine powder which was transferred to a 30 ml OakRidge tube containing 6 ml extraction buffer (7M urea, 250 mM NaCl, 50mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1% sarcosine). To this was added7 ml of phenol:chloroform 1:1, the tubes shaken and incubated at 37° C.for 15 min. Samples were centrifuged at 8K for 10 min. at 4° C. Thesupernatant was pipetted through miracloth (Calbiochem 475855) into adisposable 15 ml tube (American Scientific Products, C3920-15A)containing 1 ml 4.4 M ammonium acetate, pH 5.2. Isopropanol, 6 ml wasadded, the tubes shaken, and the samples incubated at −20°C. for 15 min.The DNA was pelleted in a Beckman TJ-6 centrifuge at the maximum speedfor 5 min. at 4° C. The supernatant was discarded and the pellet wasdissolved in 500 μl TE-10 (10 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0) 15min. at room temperature. The samples were transferred to a 1.5 mlEppendorf tube and 100 μl 4.4 M ammonium acetate, pH 5.2 and 700 μlisopropanol were added. This was incubated at −20° C. for 15 min. andthe DNA pelleted 5 min. in an Eppendorf microcentrifuge (12,000 rpm).The pellet was washed with 70% ethanol, dried, and resuspended in TE-1(10 mM Tris-HCl pH 8.0, 1 mM EDTA).

Ten μg of isolated DNA were digested with BamHI (NEB) and analyzed asdescribed in Example I using the HPT probe. As shown in FIG. 3, a bandwas observed for PH1 callus at the expected position of 1.05 Kb,indicating that the HPT coding sequence was present. No band wasobserved for control callus.

To demonstrate that the hygromycin gene is incorporated into highmolecular weight DNA, DNA isolated from PHI callus and control calluswas treated with (i) no restriction enzyme, (ii) BamHI, as describedpreviously, or (iii) PstI, which cuts the plasmid pHYGI1 only oncewithin the HPT coding sequence. Samples were blotted and probed with theHPT coding sequence as described previously.

Undigested PH1 DNA only showed hybridization to the probe at theposition of uncut DNA, demonstrating that the hygromycin gene isincorporated into high molecular weight DNA. The expected 1.05 Kb bandfor PH1 DNA digested with BamHI was observed, as had been shownpreviously. For PH1 DNA digested with PstI, a 5.9 Kb band would beexpected if the hygromycin gene was present on an intact pHYGI1 plasmid.Two or more bands of variable size (size dependent on the positionflanking PstI sites within the host DNA) would be expected if the genewas incorporated into high molecular weight DNA. Three bands wereobserved with approximate molecular sizes of 12, 5.1, and 4.9 Kb. Thisresult demonstrates incorporation of the hygromycin gene into highmolecular weight DNA. The intensity of the 4.9 Kb band is approximatelytwice as great as the other two bands, suggesting either partialdigestion or possibly a tandem repeat of the HPT gene. No hybridizationwas observed for DNA from control callus in any of the above treatments.

These results demonstrate that the HPT coding sequence is not present inPH1 callus as intact pHYGI1 or as a small non-chromosomal plasmid. Theyare consistent with incorporation of the hygromycin gene into highmolecular weight DNA. Further, Southern blot analyses demonstrated thatthe HPT coding sequence is contiguous with the 35S promoter sequence.

PH1 callus was transferred from all of the concentrations of hygromycinused in the inhibition study to RM5 medium and plants were regeneratedas described in Example I. A total of 65 plants were produced from PH1and a total of 30 plants were produced from control callus.

To demonstrate that the introduced DNA had been retained in the R0tissue, a Southern blot was performed as previously described on BamHIdigested leaf DNA from three randomly chosen R0 plants of PH1. The blotwas probed with the HPT probe as in Example I. As shown in FIG. 4, a1.05 Kb band was observed with all three plants indicating that the HPTcoding sequence was present. No band was observed for DNA from a controlplant.

Controlled pollinations of mature PH1 plants were conducted by standardtechniques with inbred Zea mays lines A188, B73, and Oh43. Seed washarvested 45 days post-pollination and allowed to dry further 1-2 weeks.

The presence of the hygromycin resistance trait in the R1 progeny wasevaluated by the root elongation bioassay, an etiolated leaf bioassay,and by Southern blotting. Two ears each from regenerated PH1 and controlplants were selected for analysis. The pollen donor was inbred line A188for all ears. The results are shown in FIG. 5 and in Table 9, below.

TABLE 9 ANALYSIS OF PH1 R1 PLANTS PH1 ROOT LEAF CONT ROOT LEAF PLANTASSAY ASSAY BLOT PLANT ASSAY ASSAY BLOT 3.1 + ND + 4.1 − ND ND 3.2 − ND− 4.2 − ND ND 3.3 − ND − 4.3 − ND ND 3.4 − ND − 4.4 − ND ND 3.5 − ND −4.5 − ND ND 3.6 + ND + 4.6 − ND ND 3.7 − ND − 4.7 − ND ND 2.1 − ND −10.1 + + + 1.1 − − − 10.2 + + + 1.2 − − ND 10.3 − − ND 1.3 − − ND 10.4 −− − 1.4 − − ND 10.5 − − − 1.5 − − ND 10.6 − − − 1.6 − − ND 10.7 − − −1.7 − − ND 10.8 ND + + 1.8 − − ND Key: + = transgenic; − =nontransgenic; ND = not done

The presence of the HPT gene was confirmed in two R2 progeny derivingfrom a PH1 maternal parent.

Plant PH1.3.1 (see Table 9) was pollinated with Oh43 pollen. Nine seedsderived from this cross were germinated, DNA was prepared from theleaves of four of the progeny plants, and analyzed by Southern blottingusing the HPT coding sequence probe. Two of the four plants testedcontained the HPT sequence in high copy number. Although the HPT genewas present in two of the plants, expression of the gene could not bedetected in the etiolated leaf assay.

Plant PH1.10.8 (Table 9) was used to pollinate B73 and was also selfed.Fifty of the progeny from the out-cross were tested for HPT expressionin both root and leaf assays. No expression was detected. LikewiseSouthern blots on DNA from eight of the progeny did not detect thepresence of the HPT gene. In the progeny from the self cross, noevidence for the presence of the gene was obtained in leaf assays ofnine progeny, or from Southern blots of the DNA from four of theseplants.

The recombinant DNA was only shoe to be inherited by progeny when atransgenic plant (both R0 and R1) was used as a female. This issuggestive of maternal inheritance of the recombinant DNA for PH1.

All of the publications and patent documents cited hereinabove areincorporated by reference herein. The invention has been described withreference to various specific and preferred embodiments and techniques.However, it should be understood that many variations and modificationsmay be made while remaining within the spirit and scope of theinvention.

What is claimed is:
 1. A fertile transgenic Zea mays plant comprising apreselected DNA sequence encoding a Bacillus thuringiensis endotoxin,wherein the preselected DNA sequence is adjusted to be more efficentlyexpressed in maize than the native B. thuringiensis DNA sequenceencoding said endotoxin, and wherein said preselected DNA is heritable.2. The transgenic Zea mays plant of claim 1 wherein the preselected DNAsequence comprises an increased G+C content of the degenerate third baseof the codons.
 3. The transgenic Zea mays plant of claim 1 or whereinthe preselected DNA sequence comprises a sequence encoding the HD73endotoxin of Bacillus thuringiensis.
 4. A seed produced by thetransgenic Zea mays plant claim 1, 3, which comprises said preselectedDNA sequence.
 5. The transgenic Zea mays plant of claim 1 wherein thepreselected DNA sequence encodes a truncated Bacillus thuringiensisendotoxin.
 6. The transgenic Zea mays plant of claim 1 wherein thepreselected DNA sequence comprises a promoter.
 7. A fertile inbredtransgenic Zea mays plant comprising a preselected DNA sequence encodinga Bacillus thuringiensis endotoxin, wherein the preselected DNA sequenceis adjusted to be more efficiently expressed in maize than the native B.thuringiensis DNA sequence encoding said endotoxin and wherein thepreselected DNA sequence is heritable.
 8. A fertile hybrid transgenicZea mays plant comprising a preselected DNA sequence encoding a Bacillusthuringiensis endotoxin, wherein the preselected DNA sequence isadjusted to be more efficiently expressed in maize than the native B.thuringiensis DNA sequence encoding said endotoxin, and wherein thepreselected DNA sequence is heritable.
 9. A fertile transgenic Zea maysplant comprising a preselected heritable DNA sequence encoding aBacillus thuringiensis endotoxin, wherein the preselected DNA sequenceis adjusted to be more efficiently expressed in maize than the native B.thuringiensis DNA sequence encoding said endotoxin, and wherein thepreselected DNA sequence further comprises a selectable marker gene or areporter gene.
 10. The transgenic Zea mays plant of claim 9 wherein thepreselected DNA sequence comprises a sequence encoding the HD73endotoxin of Bacillus thuringiensis.
 11. The transgenic Zea mays plantof claim 9 wherein the preselected DNA sequence comprises a sequenceencoding the HD1 endotoxin of Bacillus thuringiensis.
 12. A seedproduced by the transgenic Zea mays plant of claim 9, 10 or 11 whichcomprises said preselected DNA sequence.
 13. The transgenic Zea maysplant of claim 9 wherein the DNA sequence encodes a truncated Bacillusthuringiensis endotoxin.
 14. The transgenic Zea mays plant of claim 9wherein the preselected DNA sequence further comprises a promoteroperably linked to said DNA sequence encoding said endotoxin and apromoter operably linked to said selectable marker gene.
 15. A fertileinbred transgenic Zea mays plant comprising a preselected heritable DNAsequence encoding a Bacillus thuringiensis endotoxin, wherein thepreselected DNA sequence is adjusted to be more efficiently expressed inmaize than the native B. thuringiensis DNA sequence encoding saidendotoxin, and wherein the preselected DNA sequence further comprises aselectable marker gene or a reporter gene.
 16. A fertile hybridtransgenic Zea mays plant comprising a preselected heritable DNAsequence encoding a Bacillus thuringiensis endotoxin, wherein thepreselected DNA sequence is adjusted to be more efficiently expressed inmaize than the native B. thuringiensis DNA sequence encoding saidendotoxin, and wherein the preselected DNA sequence further comprises aselectable marker gene or a reporter gene.
 17. The transgenic Zea maysplant of claim 9 wherein the selectable marker gene confers resistanceor tolerance to a compound selected from the group consisting ofhygromycin, sethoxydim, haloxyfop, glyphosate, methotrexate,imidazoline, sulfonylurea, triazolopyrimidine, s-triazine, bromoxynil,phosphinothricin, kanamycin, G418, 2,2-dichloropropionic acid andneomycin.
 18. The transgenic plant of claim 17 wherein the compound isphosphinothricin.
 19. The transgenic plant of claim 17 wherein thecompound is glyphosate.
 20. The transgenic plant of claim 17 wherein thecompound is kanamycin.
 21. The transgenic plant of claim 17 wherein thecompound is hygromycin.
 22. The transgenic plant of claim 9 wherein theDNA encoding the Bacillus thuringiensis endotoxin is fused in frame withsaid selectable marker or reporter gene.
 23. The inbred transgenic plantof claim 15 wherein the DNA encodes a truncated Bacillus thuringiensisendotoxin.
 24. The hybrid transgenic plant of claim 18 wherein the DNAencodes a truncated Bacillus thuringiensis endotoxin.
 25. The transgenicplant of claim 5, 23 or 24 wherein the truncated Bacillus thuringiensisendotoxin comprises about the N-terminal 50% of the endotoxin.
 26. Thetransgenic plant of claim 1 or 9 wherein the preselected DNA furtherencodes a protease inhibitor.
 27. The transgenic plant of claim 6 or 14wherein the preselected DNA sequence further comprises the maize AdhISfirst intron or the maize Shrunken-2 first intron positioned between thepromoter and the DNA encoding said endotoxin.
 28. The transgenic plantof claim 6 or 14 wherein the preselected DNA sequence further comprisesa manopine synthase promoter, a nopaline synthase promoter or anoctopine synthase promoter.
 29. The transgenic plant of claim 6 or 14wherein the promoter is the CaMV 35S or 19S promoter.
 30. A populationof plants obtained by breeding the transgenic plants of claim 1 or 14wherein the preselected DNA sequence is transmitted by Mendelianinheritance through both male and female parent plants.
 31. An inbredinsect-resistant transgenic Zea mays plant prepared by a processcomprising: (a) crossing a fertile transgenic Zea mays plant comprisinga preselected DNA sequence encoding a Bacillus thuringiensis endotoxin,wherein the preselected DNA sequence is adjusted to be more efficientlyexpressed in maize than the a B. thuringiensis DNA sequence encodingsaid endotoxin, and wherein said DNA sequence is heritable, with amember of a second inbred Zea mays line; (b) recovering insect-resistanttransgenic progeny plants from said cross; (c) back-crossing one of thetransgenic progeny plant with a member of said second inbred line; (d)recovering insect-resistant transgenic progeny plants from said cross;and (e) repeating steps (b) and (c) to obtain said inbred plant.
 32. Theinbred transgenic Zea mays plant of claim 31 wherein said preselectedDNA sequence encodes a truncated Bacillus thuringiensis endotoxin.
 33. Atransgenic insect-resistant hybrid plant prepared by crossing the inbredplant of claim 31 or 32 with an inbred Zea mays line, and recoveringsaid hybrid plant.